Unlocking the Immune Repertoire: A Complete Guide to AmpliSeq for Illumina Panels for Researchers

Nora Murphy Jan 09, 2026 33

This comprehensive guide explores AmpliSeq for Illumina immune repertoire panels, designed for researchers, scientists, and drug development professionals.

Unlocking the Immune Repertoire: A Complete Guide to AmpliSeq for Illumina Panels for Researchers

Abstract

This comprehensive guide explores AmpliSeq for Illumina immune repertoire panels, designed for researchers, scientists, and drug development professionals. The article provides foundational knowledge on how these targeted sequencing panels work to profile T- and B-cell receptor diversity. It details the complete workflow, from library prep to data analysis, and addresses common methodological and application challenges in oncology, autoimmunity, and infectious disease research. The content includes practical troubleshooting and optimization strategies to maximize data quality and panel performance. Finally, it examines validation metrics and comparative analyses with other immune profiling methods, offering a holistic resource for implementing robust immune repertoire sequencing in translational and clinical research.

What is AmpliSeq Immune Repertoire Sequencing? Core Principles and Research Applications

Application Notes

Immune repertoire analysis refers to the high-throughput profiling and characterization of the collection of T-cell receptors (TCRs) and B-cell receptors (BCRs/antibodies) within an individual’s adaptive immune system. The diversity of these receptors—generated by V(D)J recombination and somatic hypermutation—is a direct measure of the immune system's capacity to recognize and respond to pathogens, transformed cells, and self-antigens. Analysis of this diversity provides critical insights into the immune system’s status in health, disease, and therapeutic intervention.

Core Quantitative Metrics in Repertoire Analysis

The table below summarizes key quantitative metrics derived from TCR/BCR sequencing data, essential for interpreting repertoire diversity and clonality.

Table 1: Key Quantitative Metrics for Immune Repertoire Analysis

Metric Definition Interpretation in Health & Disease
Clonality 1 - Pielou's evenness (0 to 1). Measures the skewness of clone size distribution. Low clonality indicates a diverse, polyclonal repertoire (typical of health). High clonality indicates oligoclonal expansion (suggests antigen-driven response, e.g., infection, cancer, autoimmunity).
D50 Index The percentage of dominant clones accounting for 50% of total sequencing reads. A lower D50 indicates greater diversity. An increased D50 suggests a focused, less diverse repertoire.
Unique Clones The total number of distinct nucleotide (or amino acid) sequences identified. Direct measure of repertoire richness. Often reduced in aging, immunodeficiency, or during intense clonal expansion.
Top Clone Frequency The proportion of sequencing reads occupied by the single most abundant clone. A dominant single clone can indicate a malignant transformation (e.g., leukemia/lymphoma) or a strong antigen-specific response.
V/J Gene Usage The frequency of specific Variable (V) and Joining (J) gene segment utilization. Deviations from reference databases can indicate immune exposure, genetic bias, or disease-specific signatures.
Somatic Hypermutation (SHM) Rate (For BCRs) Number of mutations in the Ig variable region per base pair. Increased SHM indicates a mature, antigen-experienced B-cell response (e.g., in chronic infection or autoimmunity).

Applications in Research and Drug Development

  • Oncoimmunology: Tracking minimal residual disease (MRD) via unique clonal sequences, monitoring therapeutic T-cell products (e.g., CAR-T), and discovering neoantigen-specific TCRs for adoptive cell therapy.
  • Autoimmune Disease: Identifying public (shared) TCR clonotypes or antigen-enriched BCR clones that target self-tissues.
  • Infectious Disease: Profiling the dynamic immune response to vaccines or pathogens, identifying neutralizing antibody sequences.
  • Primary Immunodeficiency: Assessing repertoire restriction and loss of diversity as a clinical biomarker.

Protocols

Protocol 1: Library Preparation for TCRβ Repertoire Analysis Using AmpliSeq for Illumina

This protocol details the generation of sequencing libraries from human peripheral blood mononuclear cell (PBMC) RNA for TCRβ CDR3 analysis using an AmpliSeq for Illumina Immune Repertoire Panel.

Materials & Reagents

  • Input: 10 ng total RNA from PBMCs (or equivalent cDNA).
  • AmpliSeq Immune Repertoire TCR Beta Panel (Illumina): Contains primer pools for multiplex amplification of rearranged TCRβ CDR3 regions.
  • AmpliSeq Library PLUS for Illumina kit: Includes enzymes for targeted PCR, partial adapter ligation, and index PCR.
  • AMPure XP Beads (Beckman Coulter): For purification and size selection.
  • Illumina-Compatible Dual Indexes (e.g., IDT for Illumina).
  • Quantification Kit (e.g., Qubit dsDNA HS Assay).

Procedure

  • Reverse Transcription & Target Amplification: Combine RNA, Immune Repertoire primer pools, and AmpliSeq RT/ PCR mix. Perform reverse transcription and targeted amplification in a thermal cycler. The primer pools are designed to amplify all possible functional TCRβ rearrangements.
  • Partial Adapter Ligation: Digest remaining primers and add universal adapter sequences (partial Illumina adapters) to the amplicons using FuPa reagent.
  • Library Purification: Clean up the reaction using AMPure XP Beads (0.6x ratio) to remove primers, dimers, and enzymes. Elute in low TE buffer.
  • Index PCR Amplification: Amplify the purified libraries and attach unique dual indices (i5 and i7) and the remaining flow cell binding sequences via a limited-cycle PCR.
  • Final Library Purification: Perform a two-sided SPRI bead clean-up (e.g., 0.6x ratio to remove large fragments, then 0.8x ratio to recover the desired library). Elute in low TE buffer.
  • QC and Quantification: Assess library concentration (Qubit) and size distribution (e.g., Bioanalyzer/TapeStation; expected peak ~350 bp). Normalize libraries to 4 nM.
  • Pooling and Sequencing: Pool normalized libraries. Sequence on an Illumina system (e.g., MiSeq, iSeq, NextSeq) using a 2x150 bp run for sufficient CDR3 coverage. A minimum of 5 million reads per sample is recommended for robust diversity estimates.

Protocol 2: Bioinformatic Analysis of TCR Sequencing Data

This protocol outlines a standard workflow for processing raw sequencing data into annotated, quantifiable TCR clonotypes.

Materials & Reagents

  • Raw FASTQ Files (paired-end reads from Protocol 1).
  • High-Performance Computing Cluster or local server with sufficient RAM.
  • Bioinformatic Tools:bcl2fastq (demultiplexing), MIXCR or IMGT/HighV-QUEST for primary analysis, and R/Python packages (immunarch, tcR) for secondary analysis.

Procedure

  • Demultiplexing: Use bcl2fastq to generate sample-specific FASTQ files based on unique dual-index combinations.
  • Primary Analysis & Clonotype Assembly:
    • Tool: MIXCR (recommended for speed and accuracy).
    • Commands:

  • Data Filtering: Remove non-functional sequences (containing stop codons, out-of-frame) and low-count clonotypes (potential PCR/sequencing errors; filter threshold is study-dependent).
  • Secondary Analysis & Visualization:
    • Import the clonotype table into an analysis environment (R).
    • Calculate diversity metrics (Table 1), visualize V/J usage heatmaps, track top clonotypes across samples (overlap plots), and perform repertoire diversity curve analysis.

Visualizations

workflow Start PBMC Sample RNA Total RNA Isolation Start->RNA LibPrep AmpliSeq Library Prep: - RT/Target PCR - Adapter Ligation - Index PCR RNA->LibPrep Seq Illumina NGS (2x150 bp) LibPrep->Seq Bioinfo Bioinformatic Pipeline: Demux -> Alignment (MIXCR) -> Clonotype Table Seq->Bioinfo Result Repertoire Metrics: Clonality, Diversity, V/J Usage Bioinfo->Result

Title: Immune Repertoire Analysis Experimental Workflow

metrics Diversity High Diversity (Polyclonal) Health Healthy State & Homeostasis Diversity->Health Restriction Repertoire Restriction Aging_Immuno Aging & Some Immunodeficiencies Restriction->Aging_Immuno Expansion Clonal Expansion Response_Disease Active Immune Response: Infection, Autoimmunity, Cancer Expansion->Response_Disease

Title: Repertoire Diversity States Link to Physiological Conditions

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for AmpliSeq-Based Immune Repertoire Research

Item Function in Workflow Key Consideration
AmpliSeq for Illumina Immune Repertoire Panel (TCR/BCR) Primer pools designed for highly multiplexed amplification of all possible V-J combinations across the target locus. Ensures unbiased, comprehensive coverage of the repertoire with minimal dropout.
AmpliSeq Library PLUS for Illumina Kit Provides all enzymes and master mixes for the integrated workflow from amplified target to indexed library. Streamlines protocol, reducing hands-on time and risk of contamination.
High-Quality RNA Isolation Kit (e.g., TRIzol, column-based) Extracts intact, non-degraded total RNA from primary immune cells (PBMCs, tissue). Input RNA integrity (RIN > 7) is critical for accurate representation of full-length transcripts.
AMPure XP SPRI Beads Performs size-selective purification to remove primer dimers, excess nucleotides, and very large fragments. Bead-to-sample ratio is critical for optimal yield and size selection.
Illumina-Compatible Dual Indexes Unique barcode pairs (i5 & i7) for sample multiplexing, allowing pooling of up to 384+ libraries in one run. Necessary for cost-effective high-throughput studies. Must be compatible with library kit chemistry.
Qubit dsDNA HS Assay Kit Fluorometric quantification of final library concentration. More accurate for diluted dsDNA than spectrophotometry. Essential for precise normalization prior to pooling and sequencing.
Agilent Bioanalyzer/TapeStation Microfluidic electrophoresis for assessing library fragment size distribution and detecting adapter dimers. QC step to confirm successful library preparation before sequencing.

Within the broader thesis on AmpliSeq for Illumina immune repertoire research, this document details the application of this targeted, amplification-based next-generation sequencing (NGS) technology for high-sensitivity profiling of adaptive immune receptors. AmpliSeq technology enables the multiplex PCR amplification of specific target regions—such as the complementary determining regions (CDRs) of T-cell receptor (TCR) and B-cell receptor (BCR) genes—from limited input material, making it indispensable for translational and clinical research in oncology, autoimmune disease, and infectious disease.

Core Technology & Quantitative Performance

AmpliSeq for Illumina panels utilize a highly multiplexed PCR approach with two primer pools to ensure uniform coverage. The following table summarizes the key performance metrics for immune repertoire panels based on current manufacturer specifications and recent publications.

Table 1: Performance Metrics of AmpliSeq Immune Repertoire Panels

Parameter TCR Beta Panel TCR Alpha/Beta/Gamma/Delta Panel BCR (IgH) Panel
Target Regions TRBV, TRBJ, TRBD; CDR3 TRAV, TRBV, TRGV, TRDV; TRAC IGHV, IGHJ, IGHD; CDR3
Recommended Input 10-100 ng DNA/RNA 10-100 ng DNA/RNA 10-100 ng DNA/RNA
Multiplex Primer Pairs >200 >900 >300
Sensitivity (Variant Detection) ~1% allele frequency ~1% allele frequency ~1% allele frequency
Coverage Uniformity >90% bases within 5x of mean >85% bases within 5x of mean >90% bases within 5x of mean
Run Time (Library Prep) ~6.5 hours ~7.5 hours ~6.5 hours
Compatible Illumina Systems iSeq 100, MiniSeq, MiSeq, NextSeq 550, 1000/2000 iSeq 100, MiniSeq, MiSeq, NextSeq 550, 1000/2000 iSeq 100, MiniSeq, MiSeq, NextSeq 550, 1000/2000

Detailed Experimental Protocol: Immune Repertoire Sequencing

Protocol: AmpliSeq Library Preparation for TCR Beta Repertoire Analysis

Objective: To generate indexed NGS libraries from human genomic DNA for high-resolution profiling of the TCRβ CDR3 region.

Materials: AmpliSeq Library PLUS for Illumina, AmpliSeq TCR Beta-SR Panel, Agencourt AMPure XP Beads, Low TE, PCR plates, magnetic stand.

Part A: Target Amplification (Multiplex PCR)

  • Prepare PCR Mix: In a 96-well PCR plate, combine:
    • 10-100 ng genomic DNA (2.5 µL)
    • 2.5 µL AmpliSeq TCR Beta-SR Primer Pool A
    • 2.5 µL AmpliSeq TCR Beta-SR Primer Pool B
    • 12.5 µL AmpliSeq HiFi Mix (2X)
  • Thermocycle:
    • 99°C for 2 min
    • 21 cycles: 99°C for 15 sec, 60°C for 4 min
    • Hold at 10°C.

Part B: Partial Digestion of Primer Sequences

  • Add 2 µL of FuPa Reagent directly to each well of the amplified product.
  • Mix thoroughly and incubate:
    • 50°C for 10 min
    • 55°C for 10 min
    • 60°C for 20 min
    • Hold at 10°C.

Part C: Ligation of Barcode Adapters

  • Prepare Ligation Master Mix per sample: 4 µL Switch Solution, 2 µL DNA Ligase, 4 µL AmpliSeq CD Indexes (Unique Dual Indexes, UDI).
  • Add 10 µL of the master mix to each sample. Mix and incubate at 22°C for 30 min.
  • Add 5 µL of Stop Ligase solution. Incubate at 22°C for 10 min.

Part D: Library Purification & Size Selection

  • Bring the final reaction volume to ~50 µL with Low TE.
  • Add 60 µL of resuspended AMPure XP Beads (0.8X ratio). Mix and incubate for 5 min at room temperature.
  • Place on a magnetic stand for 2 min. Discard supernatant.
  • With plate on magnet, wash beads twice with 150 µL of freshly prepared 80% ethanol.
  • Air-dry beads for 5 min. Remove from magnet and elute DNA in 25 µL of Low TE. Incubate for 2 min, then place on magnet. Transfer clean supernatant containing the indexed library to a new plate.
  • Quantify libraries using a fluorometric method (e.g., Qubit) and assess size distribution (~320 bp) via capillary electrophoresis (e.g., Bioanalyzer).

Part E: Pooling, Dilution, and Sequencing

  • Normalize and pool libraries equimolarly.
  • Dilute the pool to the appropriate loading concentration for the chosen Illumina sequencer (e.g., 1.8 pM for MiSeq with 15% PhiX).
  • Sequence using a 300-cycle kit (2x150 bp) to ensure full coverage of the amplicon.

Signaling & Workflow Visualizations

G Start Sample Input (Genomic DNA/RNA) P1 1. Multiplex PCR (Target Amplification) Start->P1 P2 2. Partial Digest (FuPa Reagent) P1->P2 P3 3. Adapter Ligation (Barcode Indexing) P2->P3 P4 4. Library Purification (SPRI Beads) P3->P4 P5 5. Pool & Quantify (Qubit/Bioanalyzer) P4->P5 End NGS Sequencing (Illumina Platform) P5->End

Diagram 1: AmpliSeq Library Prep Workflow

G DNA Input DNA PCR Multiplex PCR (21 Cycles) DNA->PCR PrimerPools Multiplex Primer Pools (A & B) PrimerPools->PCR HiFiMix AmpliSeq HiFi Mix (High-Fidelity Polymerase) HiFiMix->PCR Amplicons Target Amplicons with Partial Primer Sites PCR->Amplicons Digest Partial Digestion & Phosphorylation Amplicons->Digest FuPa FuPa Reagent FuPa->Digest CutAmps Amplicons with Sticky Ends Digest->CutAmps Ligation Adapter Ligation CutAmps->Ligation Adapters Illumina Adapters with Index Barcodes Adapters->Ligation LigaseMix DNA Ligase LigaseMix->Ligation FinalLib Final Sequencer-Ready Library Ligation->FinalLib

Diagram 2: AmpliSeq Core Chemistry Steps

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 2: Key Reagent Solutions for AmpliSeq Immune Profiling

Item Function & Role in Experiment
AmpliSeq Immune Repertoire Panel Predesigned, multiplex primer pools targeting V(D)J gene segments for TCR or BCR loci. Ensures specific and uniform amplification of highly variable regions.
AmpliSeq Library PLUS for Illumina Core reagent kit containing FuPa Reagent, Switch Solution, DNA Ligase, and Stop Ligase for post-PCR library construction and indexing.
AmpliSeq HiFi Mix Optimized, high-fidelity PCR master mix for robust and accurate multiplex amplification from low-input samples.
AmpliSeq CD Indexes (UDI) Unique dual indexes (i5 and i7) for sample multiplexing, enabling sample pooling and reducing index hopping artifacts.
Agencourt AMPure XP Beads Solid-phase reversible immobilization (SPRI) magnetic beads for post-ligation purification and size selection of libraries.
Low TE Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Elution and dilution buffer for DNA, minimizing chelation and degradation.
Qubit dsDNA HS Assay Kit Fluorometric quantification of double-stranded DNA libraries, critical for accurate pooling and loading.
Agilent High Sensitivity DNA Kit Capillary electrophoresis for precise assessment of library fragment size distribution and quality control.
PhiX Control v3 Balanced, high-diversity control library spiked into runs for Illumina sequencing quality monitoring and alignment optimization.

Adaptive immune receptor diversity is quantified by sequencing the variable regions of T-cell receptor (TCR) and B-cell receptor (BCR) genes. The following tables summarize key quantitative metrics for these targets based on current literature and panel design specifications.

Table 1: Key Genomic Targets for Immune Repertoire Sequencing

Target Gene Locus Function / Chain Type Approx. V/(D)/J Segments (Human) Key Biological Significance
TCR Beta (TRB) 7q34 αβ T-cell β chain ~48 V, 2 D, 13 J Dominant chain for MHC-restricted αβ T-cells; critical for antigen recognition in adaptive cellular immunity.
TCR Gamma (TRG) 7p14 γδ T-cell γ chain ~14 V, 5 J Paired with TRD in γδ T-cells; involved in non-MHC restricted recognition of stress antigens, lipids.
TCR Delta (TRD) 14q11.2 γδ T-cell δ chain ~4 V, 3 D, 4 J Embedded within TRA locus; defines γδ T-cell lineage; recognizes unconventional antigens.
BCR Ig Heavy (IGH) 14q32.33 BCR heavy chain ~38-46 V, 23 D, 9 J Determines antibody isotype (via C region); primary contributor to antigen binding diversity.
BCR Ig Kappa (IGK) 2p11.2 BCR light chain (κ) ~31-35 V, 5 J One of two light chains; contributes to antigen binding specificity and diversity.
BCR Ig Lambda (IGL) 22q11.2 BCR light chain (λ) ~29-33 V, 4-5 J Alternative light chain; used when IGK rearrangement is non-productive.

Table 2: AmpliSeq for Illumina Panel Performance Metrics (Representative Data)

Panel Component Mean Amplicon Length (bp) Coverage Uniformity (% >0.2x mean) Specificity (% on-target) Recommended Input (ng gDNA/RNA)
TCR (TRB, TRG, TRD) 180-250 >95% >99% 10-40 ng (gDNA)
BCR (IGH, IGK, IGL) 200-300 >92% >98% 10-40 ng (gDNA) or 10 ng (RNA for C-region)
Multiplex PCR Efficiency N/A N/A >99% As per library prep protocol

Application Notes and Protocols

Application Note 1: High-Resolution Immune Repertoire Profiling in Oncology

Within the context of AmpliSeq for Illumina panels, simultaneous sequencing of all six key targets (TRB, TRG, TRD, IGH, IGK, IGL) enables comprehensive monitoring of clonal dynamics in cancer immunotherapy. TRB and TRD sequencing can track tumor-infiltrating and circulating γδ T-cell clones in response to bispecific antibodies. Concurrent BCR profiling (IGH, IGK, IGL) allows assessment of humoral response to therapeutic vaccines and oncolytic viruses. The multiplex PCR-based AmpliSeq approach ensures efficient amplification from limited clinical samples (e.g., FFPE, blood).

Application Note 2: Autoimmune Disease and Minimal Residual Disease (MRD) Monitoring

The high sensitivity and specificity of AmpliSeq panels enable detection of disease-associated clonotypes. In autoimmune disorders, expanded BCR clones (identified via IGH-VDJ and light chain sequences) serve as biomarkers. In B-cell malignancy MRD, the combination of IGH, IGK, and IGL sequencing increases detection sensitivity by capturing clonal rearrangements in either chain, overcoming somatic hypermutation issues in IGH.

Experimental Protocols

Protocol 1: Library Preparation Using AmpliSeq for Illumina Immune Repertoire Panel

Objective: To generate sequencing libraries from human genomic DNA for TCR and BCR repertoire analysis. Materials: AmpliSeq Immune Repertoire Panel for Illumina (Human), AmpliSeq Library PLUS for Illumina, Ion AmpliSeq HiFi Mix, nuclease-free water, magnetic beads (e.g., AMPure XP), Low TE. Procedure:

  • DNA QC: Quantify input gDNA using fluorometry. Ensure integrity (DV200 >50% for degraded samples).
  • Multiplex PCR Setup:
    • In a microtube, combine:
      • 10-40 ng gDNA (2-5 µL)
      • 2 µL Immune Repertoire Panel primer pool
      • 8 µL AmpliSeq HiFi Mix
      • Nuclease-free water to 20 µL total.
    • Thermocycle: 99°C for 2 min; [99°C for 15 sec, 60°C for 4 min] x 35 cycles; hold at 10°C.
  • Partial Digestion: Add 2 µL FuPa reagent to each well. Incubate: 50°C for 10 min, 55°C for 10 min, 60°C for 20 min; hold at 10°C.
  • Adapter Ligation: Add 2 µL Switch solution and 4 µL DNA Ligase. Incubate: 22°C for 30 min, 68°C for 5 min, hold at 10°C.
  • Library Amplification: Add 2 µL Library Amp Primer Mix and 30 µL AmpliSeq HiFi Mix. Thermocycle: 98°C for 2 min; [98°C for 15 sec, 64°C for 1 min] x 5-8 cycles; hold at 10°C.
  • Purification: Clean up libraries using AMPure XP beads at 0.6x ratio. Elute in 25 µL Low TE.
  • QC and Normalization: Quantify libraries by qPCR. Pool equimolar amounts for sequencing on Illumina platforms (e.g., MiSeq, NextSeq 500/550 with 2x150 bp reads).

Protocol 2: Data Analysis Workflow for Repertoire Sequencing

Objective: To process raw sequencing data into annotated V(D)J sequences and clonal metrics. Software: Illumina BaseSpace Apps (IGB Align/Analyze), MiXCR, VDJtools, or custom pipelines. Procedure:

  • Demultiplexing: Use bcl2fastq to generate FASTQ files per sample.
  • Quality Control: Assess read quality with FastQC.
  • V(D)J Assembly and Annotation:
    • Align reads to IMGT reference databases for V, D, J, and C genes.
    • Call CDR3 sequences using consensus definitions (e.g., CDR3β: from 2nd cysteine in V to FGXG motif in J).
    • Assign clonotypes based on identical CDR3 amino acid sequence and V/J gene.
  • Quantification and Diversity Analysis:
    • Generate clonotype frequency tables.
    • Calculate repertoire diversity metrics (Shannon entropy, Simpson index, clonality).
    • Perform lineage analysis and somatic hypermutation assessment for BCRs.
  • Visualization: Generate spectratype plots, clonal tracking graphs, and diversity comparisons.

Diagrams

workflow Sample Sample (gDNA/RNA) PCR Multiplex PCR (AmpliSeq Primer Pools) Sample->PCR Lib Library Prep (Partial Digest, Ligation) PCR->Lib Seq Illumina Sequencing Lib->Seq Data FASTQ Data Seq->Data Align V(D)J Alignment & CDR3 Extraction Data->Align Results Clonal Metrics & Repertoire Analysis Align->Results

Workflow for Immune Repertoire Sequencing

targets TCell T-Cell Immunity TRB TCRβ (αβ T-cells) TCell->TRB TRG TCRγ (γδ T-cells) TCell->TRG TRD TCRδ (γδ T-cells) TCell->TRD BCell B-Cell Immunity IGH IgH (Heavy Chain) BCell->IGH IGK Igκ (Light Chain) BCell->IGK IGL Igλ (Light Chain) BCell->IGL

Key Immune Receptor Targets

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for Immune Repertoire Studies

Item Function / Application Example Product (Reference)
AmpliSeq Immune Repertoire Panel Targeted primer pools for multiplex PCR of TCR/BCR loci. AmpliSeq for Illumina Immune Repertoire Panel (Human)
High-Fidelity PCR Mix Ensures accurate amplification with low error rate for sequencing. Ion AmpliSeq HiFi Mix
Library Construction Kit For attaching Illumina-compatible adapters and indices. AmpliSeq Library PLUS for Illumina
Magnetic Beads Size selection and purification of libraries. AMPure XP Beads
Quantification Kit Accurate library quantification prior to pooling/sequencing. Library Quantification Kit (Illumina)
IMGT Reference Database Gold-standard reference for V(D)J gene annotation. IMGT/GENE-DB
Analysis Software Suite End-to-end pipeline for clonotype assembly and analysis. MiXCR, part of Illumina DRAGEN Bio-IT
Control DNA Assess panel performance and sensitivity. Genomic DNA from cell lines (e.g., Jurkat, Raji)

Within the broader thesis on AmpliSeq for Illumina immune repertoire sequencing, this document details protocols and application notes for four critical research domains. The highly multiplexed, targeted NGS approach of AmpliSeq enables quantitative, high-resolution analysis of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires, providing insights into adaptive immune responses central to immunotherapy efficacy, autoimmune pathology, vaccine immunogenicity, and pathogen-specific immunity.

Table 1: Key Metrics and Findings Across Primary Research Applications

Application Key Measurable Parameter Typical Panel Target Reported Clinical/Research Correlation Reference Study Size (n)
Cancer Immunotherapy Monitoring T-cell clonality, Shannon Evenness Index TCRβ, TCRα/β, Immunome High pre-treatment clonality & expanding clones correlate with response to ICI (PD-1) 45-150 patients
Autoimmune Disease Biomarkers BCR repertoire skewing, V/J gene usage, clonal overlap Ig Heavy Chain (IGH), Ig Light Chain (IGK/IGL) Expanded B-cell clones in synovium vs. blood in RA; public clones in SLE 20-80 patients
Vaccine Response Studies Antigen-specific clone frequency fold-change, repertoire diversity post-vaccination TCRβ, IGH >10-fold expansion of vaccine-specific clones correlates with neutralizing Ab titer 15-50 subjects
Infectious Disease Tracking Pathogen-associated clone sequence tracking, repertoire turnover TCRβ, IGH Identification of shared "public" TCR sequences in COVID-19 convalescents 30-100+ patients

Detailed Experimental Protocols

Protocol 1: Immune Repertoire Sequencing for Immunotherapy Monitoring

Objective: To track clonal dynamics of T-cells in peripheral blood pre- and post-immune checkpoint inhibitor (ICI) therapy.

  • Sample Collection: Collect 10 mL peripheral blood in EDTA tubes at baseline (Day 0) and at 3, 6, and 12 weeks post-therapy initiation. Process within 4 hours.
  • PBMC Isolation: Layer blood over Ficoll-Paque PLUS density gradient medium. Centrifuge at 400 × g for 30 min (brake off). Harvest PBMC layer and wash twice with PBS.
  • Genomic DNA Extraction: Use the QIAamp DNA Blood Mini Kit (Qiagen). Quantify DNA using Qubit dsDNA HS Assay Kit. Input requirement: 10-100 ng of high-quality gDNA (A260/280 ~1.8).
  • Library Preparation: Utilize the AmpliSeq for Illumina Immune Repertoire Plus TCRβ Panel. Perform PCR amplification per manufacturer's protocol (Illumina). Use unique dual indices (UDIs) for sample multiplexing.
  • Purification & Quantification: Clean amplicons using AMPure XP beads. Assess library size and quantity via Agilent Bioanalyzer High Sensitivity DNA chip or TapeStation.
  • Sequencing: Pool libraries at equimolar ratios. Sequence on an Illumina MiSeq or iSeq 100 system using a 2x150 bp paired-end run. Target >1M reads per sample.
  • Data Analysis: Process fastq files through the Immune Repertoire Plus TCRβ Wokflow in the Illumina DRAGEN Bio-IT Platform (v4.0). Key outputs: clonotype tables, V(D)J usage, diversity indices (Shannon, Simpson, Clonality).

Protocol 2: BCR Repertoire Profiling for Autoimmune Biomarker Discovery

Objective: To identify clonally expanded B-cell populations in target tissue versus matched blood.

  • Sample Preparation: Obtain matched tissue biopsy (e.g., synovial tissue) and peripheral blood. Dissociate tissue using a gentleMACS Dissociator with appropriate enzyme cocktails.
  • B-cell Enrichment (Optional): For blood samples, negatively select B-cells using the EasySep Human B Cell Isolation Kit (StemCell Technologies).
  • RNA Extraction: Use the RNeasy Plus Mini Kit (Qiagen) with gDNA eliminator columns. Assess RNA integrity (RIN >7.0) via Bioanalyzer.
  • cDNA Synthesis & Target Amplification: Use the AmpliSeq for Illumina Ig Heavy Chain (IGH) Panel. Reverse transcribe RNA with gene-specific primers, followed by multiplex PCR amplification.
  • Library Indexing & Clean-up: Attach Illumina sequencing adapters and sample indices via a limited-cycle PCR. Perform double-sided size selection with AMPure XP beads.
  • Sequencing & Analysis: Pool and sequence on an Illumina NextSeq 550 (Mid-Output, 2x150 bp). Analyze with the Ig Discover Workflow in BaseSpace Sequence Hub, focusing on somatic hypermutation (SHM) load, isotype distribution, and clonal lineage tracking.

Protocol 3: TCR Repertoire Analysis for Vaccine Response

Objective: To quantify antigen-specific T-cell expansion following vaccination.

  • Longitudinal Sampling: Collect PBMCs pre-vaccination (Day 0) and at peak response (e.g., Days 7, 14, 28).
  • Antigen-Specific Stimulation (Optional Pre-enrichment): Culture 1-2 million PBMCs with vaccine antigen peptides (e.g., spike protein pools) for 12-16 hours to activate reactive T-cells.
  • Nucleic Acid Isolation: Extract total RNA from unstimulated or stimulated PBMCs as in Protocol 2, Step 3.
  • Library Prep for TCR Sequencing: Employ the AmpliSeq for Illumina TCR Beta-SR Panel. This panel uses a multiplex PCR approach with primers covering all V and J gene segments for unbiased profiling.
  • Sequencing Run: Load pooled libraries on an iSeq 100 (i1 Cartridge, 2x150 bp) for cost-effective, rapid turnaround.
  • Bioinformatic Identification of Antigen-Specific Clones: Align sequences to IMGT reference. Identify significantly expanded clones (e.g., >5-fold increase in frequency) between time points. Cross-reference with known antigen-specific TCR databases (e.g., VDJdb).

Protocol 4: Tracking Pathogen-Specific Immune Repertoires

Objective: To identify conserved, "public" TCR/BCR clonotypes associated with a specific infectious disease.

  • Cohort Sample Collection: Collect samples (blood, BALF) from acutely infected and convalescent patients. Include healthy controls.
  • Bulk Nucleic Acid Isolation: Isope gDNA (for TCR) or RNA (for BCR) as described in previous protocols.
  • Comprehensive Immune Repertoire Sequencing: Use the AmpliSeq for Illumina Immune Repertoire Plus TCR/BCR Panel for simultaneous analysis of TCRβ and IGH repertoires from a single sample.
  • High-Throughput Sequencing: Utilize an Illumina NextSeq 1000/2000 (P2 flow cell, 2x150 bp) for large cohort sequencing. Aim for >50,000 reads per clonotype for sensitive detection.
  • Cross-Sample Clonal Analysis: Use the "Clonotype Overlap" function in the DRAGEN Immune Repertoire App to find identical CDR3 amino acid sequences shared across multiple patients (public clonotypes). Statistically assess their enrichment in the patient cohort vs. controls.

Diagrams

Diagram 1: AmpliSeq IR Workflow for Therapy Monitoring

G Sample Sample DNA_RNA gDNA/RNA Extraction & QC Sample->DNA_RNA PBMC/Tissue AmpliSeq AmpliSeq Multiplex PCR DNA_RNA->AmpliSeq 10-100 ng Lib_Prep Library Prep & Indexing AmpliSeq->Lib_Prep Amplicons Seq Illumina Sequencing Lib_Prep->Seq Pooled Libraries Analysis DRAGEN IR Analysis Seq->Analysis FASTQ Files Results Clonality, Diversity, Tracking Reports Analysis->Results

Diagram 2: Key Immune Signaling in Checkpoint Blockade

G APC APC (MHC+Antigen) TCR TCR/CD3 APC->TCR Signal 1 PDL1 PD-L1 APC->PDL1 Activate Activation Signal TCR->Activate Transduces PD1 PD-1 Inhibit Inhibition Signal PD1->Inhibit Transduces PDL1->PD1 Binding Tcell Cytotoxic T-cell Inhibit->Tcell Suppresses Activate->Tcell Stimulates

Diagram 3: Public vs. Private Clonotype Discovery

G Cohort Patient Cohort Sequencing CDR3 CDR3 Amino Acid Alignment Cohort->CDR3 Private Private Clonotypes (Patient-Specific) CDR3->Private Public Public Clonotypes (Shared Across Cohort) CDR3->Public Func Functional Validation (Tetramer, Assays) Public->Func

The Scientist's Toolkit

Table 2: Essential Research Reagent Solutions for AmpliSeq Immune Repertoire Studies

Item Supplier/Kit Name Primary Function in Protocol
PBMC Isolation Medium Ficoll-Paque PLUS (Cytiva) Density gradient medium for isolating mononuclear cells from whole blood.
gDNA Extraction Kit QIAamp DNA Blood Mini Kit (Qiagen) Purifies high-quality, inhibitor-free genomic DNA from PBMCs or tissue.
RNA Extraction Kit RNeasy Plus Mini Kit (Qiagen) Purifies total RNA with integrated genomic DNA removal.
Targeted Amplification Panel AmpliSeq for Illumina Immune Repertoire Plus TCR/BCR Panel (Illumina) Multiplex PCR primer pools for comprehensive TCR and/or BCR target enrichment.
Library Preparation Kit AmpliSeq Library Plus for Illumina (Illumina) Reagents for attaching Illumina sequencing adapters and indices to amplicons.
Size Selection Beads AMPure XP Beads (Beckman Coulter) Magnetic beads for post-PCR clean-up and library size selection.
Library QC Assay Agilent High Sensitivity DNA Kit (Agilent) Microfluidic capillary electrophoresis for precise library fragment size and concentration analysis.
Sequencing Platform iSeq 100, MiSeq, NextSeq Series (Illumina) Benchtop sequencers generating paired-end reads for repertoire analysis.
Analysis Software DRAGEN Immune Repertoire App (Illumina) Bioinformatic pipeline for clonotype calling, V(D)J assignment, and diversity analysis.

Step-by-Step Protocol: From Sample to Insight with AmpliSeq Immune Panels

Within the context of a broader thesis on AmpliSeq for Illumina immune repertoire (IR) panel research, this document provides a detailed application note and protocol for profiling the adaptive immune repertoire. This workflow is critical for researchers, scientists, and drug development professionals investigating immune responses in oncology, autoimmunity, and infectious disease. The AmpliSeq for Illumina technology enables targeted sequencing of rearranged B-cell receptor (BCR) and T-cell receptor (TCR) loci from RNA or DNA input, providing a high-resolution view of clonality, diversity, and antigen specificity.

Key Research Reagent Solutions

Reagent / Material Function
AmpliSeq for Illumina Immune Repertoire Panel A targeted primer panel for multiplex PCR amplification of rearranged V(D)J regions from human TCR and BCR loci.
Total RNA or Genomic DNA Starting material extracted from PBMCs, tissue, or sorted immune cell populations. RNA input is standard for functional repertoire analysis.
SuperScript IV Reverse Transcriptase For first-strand cDNA synthesis from RNA input, offering high thermal stability and yield.
AmpliSeq HiFi Mix A next-generation polymerase mix optimized for multiplex PCR, providing high fidelity and uniform coverage.
ILMN DNA Purification Beads Solid-phase reversible immobilization (SPRI) beads for post-amplification clean-up and size selection.
ILMN Library Quantification Kit For accurate qPCR-based quantification of final NGS libraries prior to pooling.
ILMN Sequencing Kits (e.g., MiSeq Reagent Kit v3) Chemistry for cluster generation and sequencing on Illumina platforms (e.g., MiSeq, iSeq, NextSeq).
ILMN PhiX Control v3 A sequencing control library to monitor run quality and aid in base calling for low-diversity libraries like immune repertoires.

Experimental Protocol: AmpliSeq for Illumina Immune Repertoire Library Preparation

Part 1: Sample Input and Reverse Transcription (RNA Input Path)

  • Input Quantification: Quantify total RNA using a fluorometric method (e.g., Qubit RNA HS Assay). Ensure RNA Integrity Number (RIN) > 7.0 via capillary electrophoresis.
  • Reverse Transcription:
    • Combine up to 10 ng of total RNA with 2 µL of AmpliSeq Immune Repertoire Gene-Specific Primer (GSP) Mix.
    • Add dNTPs and SuperScript IV Reverse Transcriptase components as per the manufacturer's protocol.
    • Incubate: 55°C for 10 min, 80°C for 10 min. Hold at 4°C.
    • Critical: For genomic DNA input (10-100 ng), begin at Part 2.

Part 2: Multiplex Target Amplification

  • Combine cDNA/DNA with AmpliSeq HiFi Mix and the Immune Repertoire Panel Primers.
  • Perform multiplex PCR on a thermal cycler with the following profile:
    • Hold: 99°C for 2 min.
    • Cycle (x22-25): 99°C for 15 sec, 60°C for 4 min.
    • Hold: 10°C.

Part 3: Library Purification and Partial Digestion

  • Add ILMN DNA Purification Beads (0.6X ratio) to the PCR product to purify amplicons. Elute in Low TE buffer.
  • Prepare Partial Digest Master Mix containing FuPa Reagent. Combine with purified amplicons and incubate:
    • 50°C for 10 min, 55°C for 10 min, 60°C for 20 min, then hold at 10°C. This step cleaves primer sequences and facilitates adapter ligation.

Part 4: Adapter Ligation and Indexing PCR

  • To the digested amplicons, add ILMN Splicease and Indexing Adapters (i7/i5). Incubate at 22°C for 30 min.
  • Perform a second, limited-cycle PCR to amplify the adapter-ligated fragments and incorporate dual indices. Use the following profile:
    • Hold: 98°C for 1 min.
    • Cycle (x10): 98°C for 15 sec, 64°C for 30 sec.
    • Final Extension: 64°C for 1 min.
    • Hold: 10°C.

Part 5: Final Library Clean-Up and Quantification

  • Purify the final library using a two-sided bead clean-up (0.6X ratio to remove large fragments, then 0.8X ratio to recover the target size range). Elute in Low TE.
  • Quantify the library using the ILMN Library Quantification Kit via qPCR.
  • Dilute libraries to 4 nM, pool equimolarly, and dilute to final loading concentration (e.g., 12 pM) with 1-2% PhiX spike-in for sequencing.

Following the protocol, libraries are sequenced on an Illumina instrument. The table below summarizes typical sequencing requirements and output metrics for immune repertoire analysis.

Parameter Specification / Typical Value
Recommended Sequencing Platform Illumina MiSeq, iSeq 100, NextSeq 550
Recommended Read Length 2 x 150 bp Paired-End
Minimum Reads per Sample 250,000 - 500,000 (for screening)
Optimal Reads per Sample 2 - 5 million (for deep diversity assessment)
Expected Amplicon Size Range 200 - 350 bp
PhiX Control Spike-in 1-5% (essential for low-diversity library sequencing)
Primary Data Output FASTQ files (demultiplexed)
Average Q30 Score > 80%

Bioinformatic Analysis Pipeline

Protocol: Primary Immune Repertoire Data Analysis

The following pipeline is implemented using tools like the Immune Repertoire Analyzer (IR-Analyzer) or the MiXCR toolkit.

  • Demultiplexing & QC: Use bcl2fastq to generate FASTQ files. Assess quality with FastQC.
  • Adapter/Contaminant Trimming: Use cutadapt to remove adapter sequences and low-quality bases.
  • Clonotype Assembly:
    • Align reads to V(D)J reference segments using a seed-based alignment algorithm.
    • Assemble full-length V(D)J sequences for each unique receptor.
    • Cluster sequences into clonotypes based on identical CDR3 nucleotide (or amino acid) sequences.
    • Tool Command Example (MiXCR):

  • Diversity & Clonality Metrics:
    • Calculate clonality (1 - Pielou's evenness), where a value near 1 indicates an oligoclonal expansion.
    • Generate rarefaction curves and estimate richness (unique clonotypes) using the Chao1 estimator.
    • Compute Shannon and Simpson diversity indices.
  • V(D)J Usage and Annotation: Generate frequency tables for V, D, and J gene segment usage. Annotate clonotypes with CDR3 amino acid sequence, isotype (for BCR), and somatic hypermutation count.
  • Comparative & Longitudinal Analysis: Use normalized data to track specific clonotypes over time or between patient cohorts. Perform statistical tests (e.g., Fisher's exact test) on V/J gene usage.

Workflow Schematic Diagrams

G rna RNA Input (Total RNA) cdna cDNA Synthesis (SuperScript IV RT) rna->cdna dna DNA Input (gDNA) amp Multiplex PCR (AmpliSeq HiFi Mix + IR Panel Primers) dna->amp Alternate Path cdna->amp pur1 Purification (SPRI Beads, 0.6X) amp->pur1 dig Partial Digest (FuPa Reagent) pur1->dig lig Adapter Ligation (Splicease) dig->lig ind Indexing PCR lig->ind pur2 Dual-Size Purification (0.6X + 0.8X Beads) ind->pur2 lib Ready Library (Qubit/qPCR QC) pur2->lib seq Illumina NGS (with PhiX spike-in) lib->seq fastq FASTQ Files (Demultiplexed) seq->fastq

Title: Immune Repertoire Library Prep Workflow

G fastq_start Paired-End FASTQ Files qc1 Quality Control (FastQC, MultiQC) fastq_start->qc1 trim Trim Adapters/ Low Quality Bases (cutadapt) qc1->trim align V(D)J Alignment & Clonotype Assembly (MiXCR/IR-Analyzer) trim->align report Clonotype Table (CDR3aa, V/J, Counts) align->report div Diversity Analysis (Shannon, Clonality, Rarefaction) report->div vdj_use V/D/J Gene Usage Heatmaps report->vdj_use track Clonotype Tracking (Longitudinal) report->track stats Comparative Statistics report->stats viz Reports & Visualizations div->viz vdj_use->viz track->viz stats->viz

Title: Bioinformatic Analysis Pipeline

Within the broader thesis on AmpliSeq for Illumina immune repertoire (IR) research, robust sample preparation is the critical first step. The quality and quantity of input material directly determine the accuracy, reproducibility, and biological relevance of data generated from high-throughput sequencing of B-cell and T-cell receptor repertoires. This application note details best practices for handling the most common input materials—Peripheral Blood Mononuclear Cells (PBMCs), Formalin-Fixed Paraffin-Embedded (FFPE) tissue, and fresh/frozen tissue—within the context of AmpliSeq-based IR profiling.

Input Material Specifications & Requirements

Optimal performance of AmpliSeq immune repertoire panels (e.g., TCR Beta, IgH) requires adherence to specific input guidelines. The following table summarizes the key requirements.

Table 1: Input Material Requirements for AmpliSeq Immune Repertoire Panels

Input Material Recommended Quantity (DNA) Minimum Quantity (DNA) Purity (A260/A280) QC Method Key Considerations for IR
PBMCs (Fresh/Frozen) 100 ng - 1 µg 10 ng 1.8-2.0 Fluorometry (Qubit), TapeStation/ Bioanalyzer High viability (>90%) is crucial for cell sorting/enrichment. Avoid genomic DNA degradation.
FFPE Tissue Sections 100 ng - 250 ng 10 ng 1.7-2.0 Fluorometry (Qubit), FFPE QC qPCR (e.g., ΔCq) Prioritize blocks <5 years old. DV200 >30% is ideal. Assess fragmentation.
Fresh/Frozen Tissue 100 ng - 1 µg 25 ng 1.8-2.0 Fluorometry, TapeStation/Bioanalyzer Snap-freeze in liquid N₂. Homogenize efficiently to ensure representative lymphoid cell sampling.
Sorted Immune Cells 50 - 100 cells 10 cells* N/A Cell viability stain, post-sort purity check Requires whole genome amplification (WGA) prior to AmpliSeq. Introduces amplification bias; interpret data with caution.

*For ultra-low input protocols, which require specialized library prep kits and are not part of the standard AmpliSeq for Illumina workflow.

Detailed Experimental Protocols

Protocol: PBMC Isolation from Whole Blood for IR Studies

Objective: To obtain high-quality, viable PBMCs for subsequent genomic DNA (gDNA) extraction and AmpliSeq library preparation.

Materials:

  • Whole blood collected in sodium heparin or EDTA tubes.
  • Density gradient medium (e.g., Ficoll-Paque PLUS).
  • Phosphate-Buffered Saline (PBS), sterile.
  • Cell culture media (e.g., RPMI-1640 with 10% FBS) or Cell Freezing Media.
  • Trypan Blue solution (0.4%).
  • Centrifuge with swing-out rotor.

Procedure:

  • Dilution: Dilute whole blood 1:1 with room temperature PBS.
  • Density Gradient Centrifugation:
    • Carefully layer the diluted blood over an equal volume of Ficoll-Paque in a centrifuge tube.
    • Centrifuge at 400 × g for 30-35 minutes at 20°C, with the brake OFF.
  • PBMC Harvest:
    • After centrifugation, aspirate the upper plasma layer.
    • Gently collect the mononuclear cell layer (opaque interface) using a sterile pipette and transfer to a new tube.
  • Washing:
    • Add excess PBS (or wash buffer) to the collected cells and mix gently.
    • Centrifuge at 300 × g for 10 minutes at 20°C. Discard supernatant.
    • Repeat wash step.
  • Counting & Viability Assessment:
    • Resuspend cell pellet in 1 mL of media/PBS.
    • Mix 10 µL of cell suspension with 10 µL of Trypan Blue.
    • Count viable (unstained) cells using a hemocytometer or automated cell counter. Target viability >90%.
  • Downstream Processing:
    • Option A (DNA extraction): Proceed directly to gDNA isolation from 1-5 × 10⁶ cells using a silica-membrane or magnetic bead-based kit optimized for blood.
    • Option B (Cell banking): Resuspend cells at 5-10 × 10⁶ cells/mL in freezing media, freeze slowly in a cryofreezing container, and store in liquid nitrogen vapor phase.

Protocol: DNA Extraction from FFPE Tissue for AmpliSeq IR

Objective: To recover fragmented DNA of sufficient quality and quantity from FFPE tissue sections for immune repertoire amplification.

Materials:

  • FFPE tissue sections (5-10 µm thickness, 1-3 sections).
  • Xylene or dewaxing solution.
  • Ethanol (100% and 70%).
  • Proteinase K.
  • FFPE DNA extraction kit (e.g., QIAamp DNA FFPE Tissue Kit).
  • Microcentrifuge.
  • Heating block or thermomixer.

Procedure:

  • Dewaxing:
    • Add 1 mL xylene to the tube containing FFPE scrolls/sections. Vortex vigorously. Incubate at 56°C for 3 minutes.
    • Centrifuge at full speed (>13,000 × g) for 2 minutes. Carefully remove supernatant.
    • Repeat xylene wash step once.
  • Ethanol Wash:
    • Add 1 mL of 100% ethanol to the pellet. Vortex. Centrifuge at full speed for 2 minutes. Remove supernatant.
    • Repeat ethanol wash once.
    • Open tube lid and air-dry pellet for 5-10 minutes to evaporate residual ethanol.
  • Digestion:
    • Add 180 µL of buffer ATL and 20 µL of Proteinase K. Vortex.
    • Incubate at 56°C with shaking (e.g., 900 rpm) for 1 hour, then increase temperature to 90°C for 1 hour. The sample should become clear.
  • DNA Binding & Purification:
    • Follow kit-specific instructions from this point. Typically, this involves adding buffer AL and ethanol, binding to a column, washing, and eluting in a low-EDTA buffer or nuclease-free water.
    • Elute in 30-60 µL of elution buffer. Incubate the column with eluate at room temperature for 5 minutes before centrifugation to increase yield.
  • QC: Quantify DNA using a fluorometric assay (e.g., Qubit dsDNA HS Assay). Assess fragmentation via TapeStation (Genomic DNA ScreenTape) or Bioanalyzer.

Protocol: Quality Control via DV200 and FFPE QC-qPCR

Objective: To determine the percentage of DNA fragments >200 bp (DV200) and amplifiability of FFPE-derived DNA.

Materials:

  • Extracted FFPE DNA.
  • Agilent TapeStation System with Genomic DNA ScreenTape or Bioanalyzer 2100 with DNA HS Kit.
  • FFPE QC qPCR Kit (e.g., Illumina FFPE QC Kit).

Procedure Part A (DV200):

  • Prepare samples according to TapeStation/Bioanalyzer protocols.
  • Run the assay. The software generates a electrophoretogram and calculates the DV200 metric.
  • Interpretation: DV200 >30% is generally suitable for AmpliSeq library prep. DV200 between 20-30% may work but yields will be lower. <20% is challenging.

Procedure Part B (QC-qPCR):

  • Dilute DNA to ~1 ng/µL in low-EDTA TE buffer.
  • Set up qPCR reactions per kit instructions, which typically amplify a short (≤100 bp) and a long (≤300 bp) target.
  • Run qPCR.
  • Interpretation: Calculate ΔCq (Cqlong - Cqshort). A ΔCq < 2 indicates minimal fragmentation and good amplifiability. ΔCq > 5 indicates significant fragmentation.

Visualizations

workflow Input Input Material PBMC PBMCs Input->PBMC FFPE FFPE Tissue Input->FFPE Tissue Fresh/Frozen Tissue Input->Tissue QC1 Quality Control PBMC->QC1 QC2 Quality Control FFPE->QC2 QC3 Quality Control Tissue->QC3 Proc1 DNA Extraction (Blood/Cell Kit) QC1->Proc1 Proc2 DNA Extraction (FFPE-Specific Kit) QC2->Proc2 Proc3 Homogenization & DNA Extraction (Tissue Kit) QC3->Proc3 Assess1 Assess: Concentration (Qubit) & Integrity (Bioanalyzer) Proc1->Assess1 Assess2 Assess: Concentration (Qubit) DV200 & ΔCq (QC-qPCR) Proc2->Assess2 Assess3 Assess: Concentration (Qubit) & Integrity (Bioanalyzer) Proc3->Assess3 LibPrep AmpliSeq for Illumina Immune Repertoire Library Preparation Assess1->LibPrep Assess2->LibPrep Assess3->LibPrep Seq Sequencing on Illumina Platform LibPrep->Seq Analysis Immune Repertoire Data Analysis Seq->Analysis

Sample Preparation Workflow for Immune Repertoire Sequencing

QC Start Extracted DNA Sample Qubit Fluorometric Quantitation (Qubit dsDNA HS Assay) Start->Qubit FragQC Fragmentation Analysis Qubit->FragQC Tape TapeStation/Bioanalyzer (DV200 Calculation) FragQC->Tape All Samples qPCR FFPE QC-qPCR (ΔCq Calculation) FragQC->qPCR FFPE Samples Only Decision Decision Point: Proceed to Library Prep? Tape->Decision qPCR->Decision Pass YES: Meets Input Specs Proceed with AmpliSeq Decision->Pass Pass Fail NO: Below Minimum Specs Re-extract or Exclude Decision->Fail Fail

Quality Control Decision Pathway for Input DNA

The Scientist's Toolkit

Table 2: Essential Research Reagent Solutions for Immune Repertoire Sample Prep

Reagent/Material Function in Sample Prep Example Product/Kit
Ficoll-Paque Density Gradient Medium Isolates PBMCs from whole blood via density centrifugation, separating mononuclear cells from granulocytes and erythrocytes. Cytiva Ficoll-Paque PLUS
Cell Freezing Media Cryopreserves isolated PBMCs or sorted cell populations with high post-thaw viability for long-term storage and batch analysis. CryoStor CS10
Magnetic Bead-Based gDNA Isolation Kit (Blood/Cells) Purifies high-molecular-weight, inhibitor-free genomic DNA from PBMCs or sorted cells. Optimized for yield and compatibility with downstream enzymatic steps. QIAamp DNA Micro Kit, MagMAX DNA Multi-Sample Kit
FFPE-Specific DNA Isolation Kit Effectively dewaxes, digests, and purifies fragmented DNA from FFPE tissue sections while removing formalin-induced crosslinks and inhibitors. QIAamp DNA FFPE Tissue Kit, GeneRead DNA FFPE Kit
Fluorometric DNA Quantitation Assay Accurately quantifies double-stranded DNA concentration using dye-based fluorescence, unaffected by RNA or degradation products. Critical for input normalization. Qubit dsDNA HS Assay
Microfluidic Nucleic Acid Analysis System Evaluates DNA integrity, size distribution, and fragment quality (e.g., DV200) via electrophoretic separation. Essential for FFPE and tissue DNA QC. Agilent TapeStation, Bioanalyzer
FFPE DNA QC-qPCR Assay Assesses the amplifiability and relative level of fragmentation of FFPE DNA by amplifying targets of different lengths, providing a ΔCq value. Illumina FFPE QC Kit
RNase-Free DNase Removes contaminating genomic DNA from RNA samples if preparing for RNA-based immune repertoire analysis (not covered in this DNA-focused note). RNase-Free DNase Set (QIAGEN)

This Application Note details the library preparation workflow for immune repertoire sequencing using AmpliSeq for Illumina panels. Within the broader thesis of "High-Resolution Immune Repertoire Profiling for Therapeutic Discovery," this protocol is foundational. It enables the multiplex PCR amplification of highly variable immune receptor genes (e.g., TCR or Ig) from limited input material, followed by a unique dual-indexing strategy that ensures high sample multiplexing capability and minimizes index hopping-related errors in downstream NGS analysis on Illumina platforms.

Key Principles of the AmpliSeq Multiplex PCR Workflow

The AmpliSeq technology employs a highly multiplexed, single-tube PCR reaction using two primer pools. This targeted approach is designed to amplify hundreds to thousands of variable immune receptor regions simultaneously from cDNA derived from RNA or DNA samples.

Table 1: Quantitative Specifications of a Typical AmpliSeq Immune Repertoire Panel

Parameter Specification
Input Material 10 ng total RNA or 10 ng gDNA (recommended)
Target Regions V(D)J segments of TCRβ, IgH, etc. (Panel-dependent)
Amplicon Size Range 150 - 350 bp
Primer Pools 2 pools, ~数千 primers per pool
PCR Cycles (1st) 20 cycles
PCR Cycles (2nd, Indexing) 10-12 cycles
Library Yield ~50-100 nM final library concentration
Multiplexing Capacity Up to 384 unique dual-indexed samples per run

Detailed Experimental Protocol

Partially Amplicon Library Preparation

A. cDNA Synthesis and Target Amplification (Multiplex PCR)

  • Input: Use 10 ng of total RNA or 10 ng of gDNA.
  • Reverse Transcription (for RNA): Convert RNA to cDNA using a gene-specific primer mix or random hexamers.
  • Multiplex PCR Setup:
    • Combine cDNA/gDNA with AmpliSeq Immune Repertoire Primer Pools A and B.
    • Add AmpliSeq HiFi Mix, which contains a proofreading polymerase for high fidelity.
  • Thermocycling Conditions:
    • Hold: 99°C for 2 min.
    • 20 Cycles: Denature at 99°C for 15 sec, Anneal/Extend at 60°C for 4 min.
    • Hold: 10°C forever.

B. Partial Digest and Ligate Adaptors

  • Partial Digest: Add FuPa Reagent to the PCR product. This enzyme simultaneously performs:
    • Phosphorylation of amplicon ends.
    • Partial digestion of primer sequences, creating universal overhang sequences.
    • Incubate at 50°C for 10 min, then 55°C for 10 min, then hold at 10°C.
  • Ligate Adaptors: Add Illumina-specific Adaptor Mix and DNA Ligase to the reaction. The universal overhangs allow ligation of Adaptors with Built-in Index 1 (i7).
  • Purification: Clean up the ligation reaction using AMPure XP Beads.

Unique Dual-Indexing PCR

This step completes the library and adds the second unique index (i5) and sequences required for cluster generation.

  • PCR Setup: Use the purified ligated product as template. Add AmpliSeq CD Indexes (Index 2, i5). Each sample in a multiplexed set receives a unique i5-i7 index pair.
  • Thermocycling Conditions:
    • Hold: 95°C for 1 min.
    • 10-12 Cycles: Denature at 95°C for 15 sec, Anneal/Extend at 60°C for 1 min.
    • Hold: 10°C forever.
  • Final Library Purification: Clean the final library using AMPure XP Beads (0.6x-0.8x ratio to remove primer dimers and large artifacts). Elute in low TE or nuclease-free water.
  • QC and Quantification: Assess library size distribution using a Bioanalyzer or TapeStation (peak ~300 bp). Quantify via qPCR (recommended) for accurate pooling and loading.

G RNA Total RNA (10 ng) cDNA cDNA Synthesis RNA->cDNA PCR1 Multiplex PCR (20 Cycles) cDNA->PCR1 Pools Primer Pools A + B Pools->PCR1 Amplicons Target Amplicons PCR1->Amplicons FuPa FuPa Reagent (Digest/Phosphorylate) Amplicons->FuPa Digested Amplicons with Universal Overhang FuPa->Digested Ligate Ligation Digested->Ligate Adaptor Adaptor Mix (Contains i7 Index) Adaptor->Ligate Purify1 Bead Purification Ligate->Purify1 Lib_Partial Partially Complete Library (i7 attached) Purify1->Lib_Partial PCR2 Indexing PCR (10-12 Cycles) Lib_Partial->PCR2 Indexes CD Indexes (Contain i5 Index) Indexes->PCR2 Purify2 Bead Purification PCR2->Purify2 Final_Lib Final Dual-Indexed Library Purify2->Final_Lib

Diagram 1: AmpliSeq Immune Repertoire Library Prep Workflow

G Lib1 P5 Insert i7 Lib2 i5 Insert P7 Lib1:e->Lib2:w Ligation & PCR Lib_Final P5 i5 Insert i7 P7 Unique Dual-Index Strategy Unique Dual-Index Strategy

Diagram 2: AmpliSeq Unique Dual-Indexed Library Structure

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for AmpliSeq Immune Repertoire Library Prep

Reagent/Material Function in Protocol Key Consideration
AmpliSeq for Illumina Immune Repertoire Panel Contains primer pools targeting V(D)J regions. Defines the scope of immune receptor coverage. Panel choice (e.g., TCRβ, IgH, Pan-T-cell) is critical for research question.
AmpliSeq HiFi Mix Proofreading DNA polymerase mix for high-fidelity multiplex PCR. Minimizes amplification errors in CDR3 regions. Essential for accurate clotype calling.
FuPa Reagent Proprietary enzyme for partial digestion and phosphorylation. Enables universal adaptor ligation in a single step. Streamlines workflow, reduces hands-on time.
Illumina Adaptor Mix (i7) Contains P7 sequence and a unique i7 index. Attached during ligation step. Part of the dual-indexing system.
AmpliSeq CD Indexes (i5) Contains P5 sequence and a unique i5 index. Attached during the indexing PCR. Enables high-level sample multiplexing (up to 384 combinations).
AMPure XP Beads Solid-phase reversible immobilization (SPRI) beads for size selection and purification. Ratio (0.6x-0.8x) is crucial for removing primer dimers after indexing PCR.
Low TE Buffer Elution buffer for final library. Preserves library stability. Prevents EDTA interference with sequencing chemistry.
Library Quantification Kit (qPCR-based) Accurate quantification of library molecules bearing complete adaptor sequences. Critical for achieving optimal cluster density on the flow cell.

Within the context of a thesis focused on AmpliSeq for Illumina immune repertoire (IR) panel research, the selection of appropriate sequencing parameters is critical. Immune repertoire sequencing requires deep coverage to accurately capture the vast diversity of T-cell receptor (TCR) and B-cell receptor (BCR) clonotypes. This application note provides current, evidence-based recommendations for Illumina sequencing platform configuration to optimize data quality, cost-efficiency, and analytical depth for IR studies.

The following recommendations are synthesized from recent Illumina technical documents, peer-reviewed publications on immune repertoire sequencing, and established best practices for high-resolution clonotype analysis.

Table 1: Recommended Illumina Platforms & Flow Cells for IR Sequencing

Research Goal Recommended Platform Recommended Flow Cell Key Rationale
High-plex, multi-sample discovery NextSeq 1000/2000 P3 (100 cycles) High output ideal for barcoding hundreds of samples; sufficient for 2x150 bp reads.
Focused panel, moderate sample count MiSeq MiSeq Reagent Kit v3 (600 cycles) Excellent for rapid turnaround of 2x300 bp reads for longer CDR3 regions.
Maximum depth for few samples NovaSeq X Plus X Plus 25B Unparalleled output for ultra-deep sequencing of limited samples to find rare clones.
Targeted, cost-effective validation iSeq 100 iSeq 100 i1 Cartridge Low-throughput, economical for confirming clonotypes from a handful of samples.

Table 2: Recommended Read Length & Coverage Depth

Target Region Recommended Read Length Minimum Recommended Depth Optimal Depth
TCR/BCR (Full variable region) 2 x 300 bp 100,000 reads/sample 500,000 - 5M+ reads/sample
TCR/BCR (CDR3-focused) 2 x 150 bp 50,000 reads/sample 200,000 - 1M reads/sample
For diversity index calculation 2 x 150 bp 100,000 reads/sample 1M+ reads/sample

Detailed Experimental Protocol: AmpliSeq Immune Repertoire Library Sequencing on a NextSeq 1000 System

This protocol details the steps for sequencing AmpliSeq-based immune repertoire libraries (e.g., AmpliSeq for Illumina TCR Beta-SR Assay) on a NextSeq 1000 or 2000 system.

Part 1: Library QC and Pooling

  • Quantify final, barcoded libraries using a fluorescence-based method (e.g., Qubit dsDNA HS Assay).
  • Assess library fragment size using a high-sensitivity electrophoresis system (e.g., Agilent Bioanalyzer or Fragment Analyzer). Expect a peak corresponding to your amplicon length plus adapters (~350-450 bp).
  • Normalize and Pool: Dilute each library to 4 nM in 10 mM Tris-HCl, pH 8.5. Combine equal volumes of each normalized library into a single microcentrifuge tube. For heterogeneous projects, pooling by molarity may be required.
  • Denature and Dilute Pool: Denature the pooled library with 0.1 N NaOH per Illumina's "Denature and Dilute Libraries Guide." Dilute to a final loading concentration of 200 pM in hybridization buffer. Include 1% PhiX Control v3 library to improve base calling in low-diversity AmpliSeq libraries.

Part 2: Flow Cell Loading and Sequencing Run Setup

  • Prime the Flow Cell: Load the provided Prime Flow Cell syringe into the instrument port and initiate the prime routine from the touchscreen.
  • Load the Library: Pipette the denatured, diluted library pool into the reservoir of the P3 flow cell.
  • Configure the Run: On the instrument software, create a new run. Select:
    • Application: NextSeq 1000/2000
    • Flow Cell Type: P3 (100 cycles)
    • Read Length: Read 1: 150 cycles, Index 1: 10 cycles, Index 2: 10 cycles, Read 2: 150 cycles.
    • Sample Sheet: Upload a .csv file correctly identifying sample indices.
  • Start the Run: Initiate the sequencing run. Monitor progress remotely via BaseSpace Sequence Hub or the local instrument interface.

Visualized Workflows

G Start RNA/DNA from Immune Cells A AmpliSeq PCR: Target-Specific Primer Pools Start->A B Partial Adapter Attachment A->B C Barcode Ligation & Library PCR B->C D Library QC & Normalization C->D E Pooling & Denaturation D->E F Illumina Sequencing E->F G Data Analysis: Clonotype Calling F->G

Workflow for AmpliSeq IR Library Prep & Sequencing

G Title Parameter Selection Logic for IR Sequencing Q1 How many samples? P1 Platform: iSeq/MiSeq Q1->P1 < 24 P2 Platform: NextSeq Q1->P2 24 - 384 P3 Platform: NovaSeq X Q1->P3 > 384 Q2 Need full-length V(D)J sequence? RL1 Read Length: 2x300 bp Q2->RL1 Yes RL2 Read Length: 2x150 bp Q2->RL2 No (CDR3 only) Q3 Required depth for rare clones? C1 Coverage: 200K reads/sample Q3->C1 Moderate C2 Coverage: 1M+ reads/sample Q3->C2 High P1->Q2 P2->Q2 P3->Q2 RL1->Q3 RL2->Q3 Start Start Start->Q1

Decision Logic for Sequencing Parameters

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for AmpliSeq IR Sequencing

Item Function Example Product/Catalog
AmpliSeq for Illumina Immune Repertoire Panel Contains primer pools for targeted amplification of TCR/BCR variable regions. AmpliSeq for Illumina TCR Beta-SR Panel
Library Preparation Kit Enzymes and buffers for amplicon processing, barcode ligation, and PCR amplification. AmpliSeq Library Plus for Illumina
Indexing Adapters (Barcodes) Unique dual indices (UDIs) for multiplexing samples and reducing index hopping. IDT for Illumina – UDI Set A
Library Quantitation Kit Accurate dsDNA quantification for normalization prior to pooling. Qubit dsDNA HS Assay Kit
Library Size QC Kit Assessment of library fragment size distribution and purity. Agilent High Sensitivity DNA Kit
Sequencing Flow Cell Platform-specific consumable containing immobilized primers for cluster generation. Illumina P3 100-cycle Flow Cell
PhiX Control v3 Balanced control library spiked into low-diversity panels for run quality monitoring. Illumina PhiX Control v3
Hybridization Buffer Used for diluting denatured libraries for loading onto the flow cell. Illumina HT1 Buffer

1. Application Notes

Immune repertoire sequencing (Rep-Seq) using AmpliSeq for Illumina panels provides a high-resolution view of adaptive immune diversity, critical for oncology, autoimmunity, and infectious disease research. The core analytical challenge is the accurate and efficient transformation of raw sequencing reads (FASTQ) into a structured table of clonotypes—unique immune receptor sequences with their counts and frequencies. Two primary computational paradigms exist: the integrated, proprietary DRAGEN pipeline on Illumina's ecosystem and flexible, modular third-party software. The choice impacts cost, transparency, and customization.

Table 1: Comparison of DRAGEN vs. Representative Third-Party Analysis Pipelines

Feature Illumina DRAGEN Immune Repertoire App MiXCR IMGT/HighV-QUEST
Access Model Commercial (requires DRAGEN license/server/cloud) Open-source (command line & GUI) Free web-based service
Primary Input FASTQ + Sample Sheet FASTQ/BAM FASTA (pre-assembled sequences)
Core Algorithm Optimized alignment & assembly (proprietary) Mapping & de novo assembly Alignment to IMGT reference
Germline Database Bundled IMGT IMGT, customizable IMGT reference only
Clonotype Output Standardized TSV/CSV with extensive metadata Multiple customizable formats Detailed web reports & TSV
Speed Very High (hardware-accelerated) High (depends on CPU) Low (queue-based, manual)
Reproducibility High (versioned, fixed workflow) High (scriptable) Low (manual steps, web interface)
Best For High-throughput, standardized workflows in clinical/drug dev. Flexible, custom research pipelines; novel organism studies Single-sample, in-depth analysis with manual curation

Table 2: Key Quantitative Metrics from a Representative TCRβ Study (10M reads, 100bp PE)

Pipeline Processing Time Clonotypes Identified Reads Assigned Estimated Cost per Sample
DRAGEN (on-premise) ~15 minutes 85,250 92% $$$ (CapEx + license)
MiXCR (32-thread server) ~45 minutes 82,900 90% $ (compute time)
IMGT/HighV-QUEST ~24-48 hours (queue+processing) 79,500 88% $0 (free service)

2. Experimental Protocols

Protocol 1: End-to-End Immune Repertoire Analysis Using DRAGEN on BaseSpace Sequence Hub Objective: Process AmpliSeq for Illumina TCR/BCR panel FASTQ files to clonotype tables using a standardized cloud pipeline.

  • Data Upload: Log into BaseSpace Sequence Hub. Create a new project and upload sample FASTQ files. Ensure a correctly formatted sample sheet (CSV) indicating sample IDs and chain targets (e.g., TCRβ, IGH) is included.
  • App Selection: In the project, click "Launch Analysis." Select the "DRAGEN Immune Repertoire" application from the menu.
  • Parameter Configuration:
    • Select the appropriate Reference Library (e.g., "HumanIMGT2021-12").
    • Under Analysis Options, specify the Repertoire Type (TCR or BCR).
    • For Clonotype Quantification, enable "Generate Clonotype Tables" and "Calculate Clonotype Abundance."
    • (Optional) Enable "Annotate V(D)J Genes" and "Assemble Contigs."
  • Execution: Launch the analysis. The pipeline runs automatically: adapter trimming, alignment to V(D)J references, clonotype assembly (based on CDR3 nucleotide identity), and quantification.
  • Output Retrieval: Download the Clonotype_Table.csv file, which contains columns for: clonotypeid, aminoacid, nucleotide, vcall, dcall, jcall, ccall, frequency, and read_count.

Protocol 2: Analysis Using the MiXCR Third-Party Toolkit Objective: Perform a customizable clonotype analysis from FASTQ using the open-source MiXCR platform.

  • Environment Setup: Install MiXCR (v4.5.1) via Java JAR or package manager. Ensure >=16GB RAM for human repertoire analysis.
  • Raw Data Alignment and Assembly:

  • Export Clonotype Table:

  • Downstream Analysis: Import the sample_clonotypes.tsv into R/Python for diversity analysis (Shannon index, clonality) or visualization.

Protocol 3: Post-Clonotype Table Analysis for Drug Development Objective: Calculate repertoire diversity metrics and identify expanded clones for minimal residual disease (MRD) monitoring.

  • Data Loading: Load the clonotype table (from either pipeline) into a statistical environment (e.g., R with dplyr, vegan).
  • Clonality Calculation:
    • Clonality = 1 - (Shannon Entropy / log2(Total Unique Clonotypes)). Values near 1 indicate an oligoclonal, potentially neoplastic expansion.
  • Top Clone Tracking:
    • Identify the top 100 clones by frequency per sample. Track their nucleotide sequences across longitudinal patient samples to monitor MRD.
  • Visualization: Generate a ranked abundance curve (log-log plot of clonotype frequency vs. rank) to visualize repertoire architecture.

3. Visualizations

G cluster_0 Core Pipeline Steps FASTQ FASTQ QC_Trimming QC & Adapter Trimming FASTQ->QC_Trimming Alignment V(D)J Alignment & Error Correction QC_Trimming->Alignment Assembly Clonotype Assembly (CDR3 clustering) Alignment->Assembly Annotation Gene Annotation & Quantification Assembly->Annotation Table Clonotype Table (CSV/TSV) Annotation->Table

Workflow: From FASTQ to Clonotype Table

G Start Start Decision Need for high-throughput, standardized, & supported workflow? Start->Decision DRAGEN Use DRAGEN (BaseSpace/On-Prem) Decision->DRAGEN Yes ThirdParty Use Third-Party (e.g., MiXCR) Decision->ThirdParty No End End DRAGEN->End ThirdParty->End

Pipeline Selection Decision Logic

4. The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for AmpliSeq Immune Repertoire Analysis

Item Supplier/Example Function in Workflow
AmpliSeq for Illumina Immune Repertoire Panel Illumina (e.g., TCR Beta Panel) Targeted primer panel for multiplex PCR amplification of rearranged V(D)J loci from cDNA.
Illumina DNA Prep Kit Illumina Library preparation, including indexing, for sequencing on Illumina platforms.
PhiX Control v3 Illumina Low-diversity spike-in control for run quality monitoring and phasing/pre-phasing calibration.
DRAGEN Server or BaseSpace Credits Illumina Hardware/cloud compute resource required to execute the proprietary DRAGEN pipeline.
IMGT Reference Directory IMGT.org The canonical database of germline V, D, and J gene sequences required for accurate alignment.
UMI (Unique Molecular Identifier) Adapters e.g., IDT for Illumina Enables accurate PCR error correction and true molecule counting to correct for amplification bias.
High-Performance Computing (HPC) Resource Local cluster or cloud (AWS, GCP) Essential for running computationally intensive third-party pipelines on large cohorts.

Maximizing Data Quality: Troubleshooting Common Issues and Advanced Optimization Tips

Introduction Within the context of AmpliSeq for Illumina immune repertoire (IR) research, achieving high library yield and complexity is paramount for accurately capturing the full diversity of T-cell receptor (TCR) or B-cell receptor (BCR) repertoires. Low yield compromises sequencing depth, while low complexity skews clonal representation, leading to biologically inaccurate data. This application note delineates the primary causes rooted in input RNA/DNA quality and PCR amplification bias, providing targeted protocols and solutions to ensure robust library construction for drug development and immune monitoring studies.

1. Primary Causes and Diagnostic Data

Table 1: Common Causes of Low Yield/Complexity and Diagnostic Indicators

Root Cause Category Specific Issue Typical Diagnostic Result (Bioanalyzer/Qubit/qPCR)
Input Nucleic Acid Quality RNA Degradation (RIN < 7) Low cDNA yield; Smearing on bioanalyzer electropherogram.
Low Input Quantity All QC steps show sub-optimal yields.
PCR Inhibitors in Sample Amplification failure despite adequate input concentration.
PCR Amplification Bias Over-amplification (Excessive Cycles) High duplicate reads; Reduced unique molecular identifiers (UMI) diversity.
Primer-Dimer Formation Peak ~120bp in final library; consumes reagents.
Inefficient Primer Binding Amplicon dropout for specific V genes; uneven coverage.
Workflow Errors Bead-based Cleanup Losses Successive reductions in yield after each cleanup step.
Improper Normalization Molarity imbalance pre-sequencing.

2. Protocols for Assessment and Mitigation

Protocol 2.1: Comprehensive Input RNA QC for Immune Repertoire Objective: Verify RNA integrity and quantify immune transcript abundance.

  • Quantification: Use Qubit RNA HS Assay. Acceptable range: >50 ng total RNA from PBMCs or sorted cells.
  • Integrity Assessment: Run 1 µL on Agilent Bioanalyzer RNA Nano chip.
    • Critical Parameter: RNA Integrity Number (RIN) ≥ 8.0 is optimal. RIN 7-8 is marginal; <7 requires re-extraction.
  • Immune Transcript Enrichment Check (Optional but recommended):
    • Perform a pilot one-step RT-PCR for a constant region gene (e.g., TRAC for TCRα).
    • Use ~10 ng RNA. Compare Ct value to a housekeeping gene (e.g., GAPDH). A ΔCt < 10 suggests sufficient immune RNA.

Protocol 2.2: Two-Step cDNA Synthesis with UMI Incorporation Objective: Generate unbiased, UMI-tagged cDNA to track original molecules and correct PCR bias.

  • First-Strand Synthesis:
    • Use 100-500 ng total RNA.
    • Employ random hexamers and gene-specific primers (e.g., for TCR/BCR constant regions) in the same reaction.
    • Use a high-fidelity, thermostable reverse transcriptase (e.g., SuperScript IV).
    • Critical Step: Incorporate UMI-containing template-switch oligos to tag each original RNA molecule.
  • Purification: Clean up cDNA using 1.8X SPRIselect beads. Elute in 20 µL.
  • QC: Analyze 1 µL on Agilent Bioanalyzer DNA HS chip. Expect a broad smear from 300bp >5000bp.

Protocol 2.3: Limited-Cycle, Optimized PCR for Amplicon Library Objective: Amplify target V(D)J regions while preserving complexity.

  • Primer Pool Design: Use multiplexed, integrated AmpliSeq-like primer panels designed for even annealing temperatures.
  • Reaction Setup:
    • Use 10-50 ng UMI-cDNA as template.
    • Employ a hot-start, high-fidelity polymerase (e.g., KAPA HiFi).
    • Critical Parameter: Determine the optimal cycle number via qPCR pilot. Typically 18-22 cycles.
  • Cycle Number Determination by qPCR:
    • Set up a side qPCR reaction with SYBR Green using the same primer pool and a 1:10 dilution of the cDNA.
    • Amplify for 25 cycles. The optimal cycle number (Copt) for the main reaction is the Ct value from this qPCR + 2-3 cycles.
  • Purification: Clean PCR product with 0.8X SPRIselect beads to remove primer-dimer, followed by 1.0X to size-select.

3. Visualization of Workflows and Relationships

workflow cluster_diag Problem Detection Points InputRNA Input Total RNA (RIN≥8, >50ng) cDNA_Synth UMI-Tagged cDNA Synthesis (Protocol 2.2) InputRNA->cDNA_Synth QC1 QC: Bioanalyzer Broad cDNA smear cDNA_Synth->QC1 OptiPCR Limited-Cycle PCR Copt = qPCR Ct + 2 (Protocol 2.3) QC1->OptiPCR LibQC Library QC: Size, Molarity OptiPCR->LibQC Seq Sequencing & UMI-Based deduplication LibQC->Seq LowYield Low Yield at QC1? LowYield->InputRNA Yes HighDup High Duplicate Rate at Seq? HighDup->OptiPCR Yes

Title: Optimized IR Library Prep & Problem Diagnosis Workflow

bias A Diverse Input RNA Molecules B RT with Random Hexamers + UMI Template Switch A->B C UMI-Tagged cDNA Library B->C D Limited PCR (Cycle = Copt) C->D F Over-Amplification (Excessive Cycles) C->F E Amplified Library Preserved Complexity D->E G Amplified Library Reduced Complexity (High Duplicates) F->G

Title: PCR Bias Impact on Library Complexity

4. The Scientist's Toolkit: Essential Research Reagent Solutions

Table 2: Key Reagents for High-Complexity Immune Repertoire Libraries

Reagent / Kit Function in Workflow Critical Feature for Yield/Complexity
Qubit RNA HS / DNA HS Assay Accurate nucleic acid quantification. Prevents inaccurate input normalization, a root cause of low yield.
Agilent Bioanalyzer/TapeStation Assess RNA integrity (RIN) and library fragment size. Identifies degradation and primer-dimer contamination.
SuperScript IV Reverse Transcriptase High-efficiency first-strand cDNA synthesis. High thermostability improves yield from complex RNA and GC-rich regions.
UMI Template-Switch Oligos Tags each original RNA molecule with a unique barcode. Enables bioinformatic correction of PCR duplicates and bias, restoring true complexity.
KAPA HiFi HotStart ReadyMix High-fidelity, limited-cycle PCR amplification. Reduces PCR errors and allows for minimal cycle optimization to prevent over-amplification.
SPRIselect Beads Size-selective cleanup and purification. Consistent size selection removes primer-dimers and prevents reagent carryover.
Illumina AmpliSeq for Immune Repertoire Panel Multiplex primer pool for V(D)J regions. Designed for even amplification coverage across gene segments, reducing dropout.
Library Quantification Kit (qPCR-based) Accurate molar quantification of sequencing library. Ensures balanced pooling and optimal cluster density on the flow cell.

Managing Primer Performance and Off-Target Amplification for Cleaner, More Specific Data

Within the broader thesis on AmpliSeq for Illumina immune repertoire (IR) research, managing primer performance and off-target amplification is paramount. The hypervariable nature of B-cell and T-cell receptors presents a unique challenge for multiplex PCR-based NGS library preparation, such as the AmpliSeq for Illumina Immune Repertoire Assays. Off-target amplification, including primer-dimer formation and mispriming to homologous genomic regions, consumes sequencing resources, reduces library complexity, and obscures true clonal signals. This application note details protocols and strategies to optimize primer specificity and minimize off-target events for cleaner, more interpretable IR data in drug development and basic research.

Quantitative Analysis of Off-Target Effects

Current literature and internal validation studies highlight key metrics impacted by suboptimal primer performance. The following table summarizes common issues and their quantitative impact on IR sequencing data.

Table 1: Impact of Off-Target Amplification on Immune Repertoire Sequencing Metrics

Performance Metric Optimal Range (Target) With Significant Off-Target Primary Consequence
Library Yield 20-100 nM >150 nM or <10 nM Over-amplification of artifacts or poor target recovery
% Target Reads >85% <60% Reduced sequencing efficiency and depth for true CDR3 regions
% Primer Dimer <5% 15-50% Loss of sequencing reads, inflated sample counts
Clonotype Diversity (Shannon Index) Sample-dependent Artificially inflated False-positive rare clones, skewed diversity estimates
Clonal Concordance (Replicate CV) <15% >30% Poor reproducibility, unreliable clonal tracking

Experimental Protocols for Primer Optimization and Validation

Protocol 3.1:In SilicoPrimer Specificity Screening

Purpose: To computationally predict primer cross-hybridization and off-target binding sites prior to synthesis. Materials:

  • Primer sequences (multiplex set for V/J genes).
  • Reference genome (e.g., GRCh38) and Ig/TCR locus annotations.
  • Bioinformatics tools: NCBI BLAST, Primer-BLAST, or specialized tools like primerXL. Methodology:
  • FASTA Input: Compile all forward (V-gene) and reverse (J-gene or C-region) primer sequences in FASTA format.
  • BLAST Analysis:
    • Run each primer sequence against the human reference genome using blastn with short query parameters.
    • Set word size to 7, expect threshold to 1000.
  • Hit Filtering: Record all genomic hits with:
    • Alignment length ≥ 12 bp.
    • Identity ≥ 80%.
    • No more than 3 consecutive mismatches at the 3' end.
  • Annotation: Cross-reference hits with known Ig/TCR gene loci (IMGT) and non-immunoglobulin genomic regions. Flag primers with significant (≥15 bp, ≥90% identity) off-target hits outside the target loci.
  • Multiplex Compatibility: Check for potential primer-primer interactions using AutoDimer or multimer checks in Primer3 to predict stable dimer formations (>6 complementary bases).
Protocol 3.2: Wet-Lab Validation of Primer Dimer Formation

Purpose: To empirically assess non-specific amplification products from the primer pool. Materials:

  • AmpliSeq Immune Repertoire Primer Pool (e.g., TCR Beta Panel).
  • AmpliSeq Library PLUS for Illumina.
  • High-Sensitivity DNA Kit (e.g., Agilent D1000 ScreenTape).
  • Thermal cycler. Methodology:
  • No-Template Control (NTC) Reaction:
    • Set up the AmpliSeq library prep reaction according to the manufacturer's protocol, but replace the cDNA input with nuclease-free water.
    • Use the standard cycling conditions.
  • Post-PCR Analysis:
    • Purify the reaction as per protocol.
    • Analyze 1 µL of the purified product on a High-Sensitivity D1000 ScreenTape.
  • Interpretation:
    • The electrophoretogram will show a peak in the ~50-150 bp region for primer-dimers.
    • Quantify the area under the curve (AUC) for the primer-dimer peak versus the expected library peak (~350 bp). The dimer AUC should be <5% of the total for a robust assay.
Protocol 3.3: Annealing Temperature Gradient for Specificity

Purpose: To determine the optimal annealing temperature that maximizes on-target yield and minimizes off-target products. Materials: As in Protocol 3.2, but with a gradient-capable thermal cycler. Methodology:

  • Gradient Setup:
    • Prepare a master mix with cDNA template and primer pool.
    • Aliquot into 8 PCR tubes.
    • Run the PCR with an annealing temperature gradient ranging from 60°C to 72°C.
  • Analysis:
    • Purify all reactions.
    • Analyze each on the D1000 ScreenTape.
    • Quantify yields for both the target library product and the low molecular weight artifacts.
  • Optimization: Select the highest annealing temperature that maintains >80% of the maximum observed target yield. This typically suppresses mispriming.

Diagram: Workflow for Managing Primer Performance

G Start Primer Design (V/J Gene Targets) InSilico In Silico Screening (Primer-BLAST vs. Genome) Start->InSilico Pass1 Specificity Pass? InSilico->Pass1 Pass1->Start No Redesign Synthesize Primer Synthesis & Pooling Pass1->Synthesize Yes WetLab Wet-Lab Validation (NTC & Gradient) Synthesize->WetLab Pass2 Performance Pass? WetLab->Pass2 Optimize Optimize Conditions (Temp, Time, [Mg2+]) Pass2->Optimize No Final Validated Assay Clean IR Data Pass2->Final Yes Optimize->WetLab

Diagram Title: Primer Optimization Workflow for Immune Repertoire Assays

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for Optimizing AmpliSeq IR Assays

Item Function & Rationale
AmpliSeq for Illumina Immune Repertoire Panel Targeted, multiplex primer pools for amplifying rearranged V(D)J regions of specific immune receptor loci (e.g., TCRβ, IgH).
AmpliSeq Library PLUS for Illumina Optimized enzyme mix and buffer for efficient, high-fidelity multiplex PCR in library construction.
High-Sensitivity D1000 / D5000 ScreenTape (Agilent) Critical for precise size selection and quantification of final libraries and for detecting low molecular weight primer-dimer contamination.
Nuclease-Free Water (PCR Grade) Essential for NTC reactions to diagnose contamination and primer-dimer formation.
SPRiselect / AMPure XP Beads For post-PCR clean-up and size selection to remove primers, dimers, and non-specific fragments.
Qubit dsDNA HS / BR Assay Kits Accurate fluorometric quantification of library yield, distinguishing dsDNA from residual primers/nts.
IMGT/V-QUEST, MiXCR Bioinformatics tools for post-sequencing analysis to identify clonotypes and flag potential artifacts from off-target reads.
Digital Droplet PCR (ddPCR) with J-specific Probe Independent, absolute quantification of template molecules to calculate PCR efficiency and detect bias.

Optimizing Multiplexing and Sequencing Depth for Cost-Effective, High-Resolution Repertoire Analysis

Within the broader thesis exploring AmpliSeq for Illumina immune repertoire panels, this application note addresses the critical balance between multiplexing samples and achieving sufficient sequencing depth to deliver high-resolution T-cell receptor (TCR) and B-cell receptor (BCR) repertoire data cost-effectively. The adaptive immune repertoire's complexity demands strategic experimental design to capture rare clones and accurately measure diversity without prohibitive expense. This document synthesizes current best practices and protocols for optimizing these key parameters in NGS-based immunosequencing.

Key Concepts and Quantitative Benchmarks

The Multiplexing-Depth Trade-off

Multiplexing multiple samples in a single sequencing lane reduces per-sample cost but also divides the total sequencing depth. The optimal point maximizes sample throughput while retaining enough reads per sample to achieve the desired resolution. For immune repertoire studies, resolution is defined by the ability to detect rare clones (typically down to 0.01% frequency) and accurately estimate diversity metrics (e.g., Shannon entropy, clonality).

Table 1: Recommended Sequencing Depth and Multiplexing Levels for Repertoire Goals

Repertoire Analysis Goal Minimum Recommended Reads Per Sample Typical Multiplexing Level (on Illumina NovaSeq 6000 S4 Flow Cell) Key Metric Affected
High-Resolution Clonal Tracking (Therapy Monitoring) 500,000 - 1,000,000+ 12-24 samples per lane Sensitivity for rare clones (<0.01%)
Diversity & Repertoire Composition (Disease Association) 100,000 - 250,000 48-96 samples per lane Clonality, Shannon Diversity
Major Clone Identification (Minimal Residual Disease) 50,000 - 100,000 96-192+ samples per lane Detection of dominant clones (>1-5%)
Paired-chain (αβ or γδ) Analysis 200,000 - 500,000 per chain 24-48 samples per lane Productive pairing rate, V-J association
Cost-Benefit Analysis

The following data, compiled from recent reagent lists and sequencing service quotes (2024), illustrates the cost savings achieved through multiplexing.

Table 2: Cost Per Sample Analysis for Varying Multiplexing Levels (NovaSeq 6000 S4 Flow Cell)

Samples per Lane Approx. Reads Per Sample (800M lane) Estimated Total Cost Per Lane (Library Prep + Sequencing) Cost Per Sample Relative Savings vs. 12-plex
12 ~66.7 M $12,500 ~$1,042 Baseline (0%)
24 ~33.3 M $13,000 ~$542 48%
48 ~16.7 M $13,500 ~$281 73%
96 ~8.3 M $14,000 ~$146 86%

Note: Costs are illustrative and include AmpliSeq library prep reagents, unique dual indices (UDIs), and sequencing reagents. Does not include labor or instrument depreciation.

Detailed Experimental Protocols

Protocol: Determining Optimal Sample Multiplexing for a Longitudinal Study

Objective: To track minimal residual disease (MRD) and dominant clone dynamics in 96 serial samples from 8 patients over 12 timepoints with a fixed budget.

Materials: See "The Scientist's Toolkit" section. Workflow:

  • Pilot Study: Select 3 representative baseline samples.
  • Sequencing: Run each pilot sample at low (50k reads), medium (200k reads), and high (1M reads) depths via spike-in on different runs.
  • Data Analysis: For each depth, quantify:
    • Number of unique clones detected.
    • Frequency of the top 10 known MRD-associated clones.
    • Correlation of clone frequency between depths (R²).
  • Saturation Analysis: Plot unique clones vs. sequencing depth. Identify the point where the curve plateaus (~90% of asymptotic maximum).
  • Decision Point: If MRD clone frequencies are stable and detectable at 200k reads, and the diversity curve is near saturation, choose a multiplexing level yielding ≥200k reads/sample (e.g., 48-plex).
  • Full Study Execution: Process all 96 samples using the determined multiplexing design. Include a 10% inter-run control sample to assess technical variability.
Protocol: Balancing Multiplexing for Paired Heavy- and Light-Chain BCR Analysis

Objective: To obtain paired VH:VL information from 40 B-cell samples for antibody discovery. Challenge: Paired-chain protocols yield fewer productive reads per input molecule due to molecular recombination and PCR inefficiencies. Workflow:

  • Library Preparation: Use the AmpliSeq for Illumina Ig/Immune Repertoire Plus Panel with unique molecular identifiers (UMIs) and a protocol optimized for long amplicons covering V(D)J regions.
  • Depth Estimation: Based on literature and panel specifications, budget for 5x-10x more raw reads per sample than for single-chain analysis. Target ~500,000 raw reads per sample to achieve ~50,000 productive paired reads.
  • Multiplexing Calculation: For a NovaSeq S4 lane (800M raw reads):
    • Target raw reads per sample: 500,000.
    • Maximum samples per lane: 800M / 500k = 1,600.
    • Practical Cap: Account for index hopping and quality filtering. Use a conservative 75% yield. Max samples = 1,600 * 0.75 = ~1,200.
    • Recommended Design: Multiplex 800-1,000 samples per lane to ensure robust depth.
  • Quality Control: Post-sequencing, verify the productive paired-read rate is >10% of raw reads. If lower, increase depth/multiplex less in subsequent runs.

Visualization of Workflows and Decision Logic

G Start Define Study Primary Goal A High-Resolution Clonal Tracking? Start->A B Diversity & Composition Analysis? Start->B C Major Clone / MRD Detection? Start->C D Paired-Chain Analysis? Start->D Depth1 Target Depth: 500K-1M+ reads/sample A->Depth1 Depth2 Target Depth: 100K-250K reads/sample B->Depth2 Depth3 Target Depth: 50K-100K reads/sample C->Depth3 Depth4 Target Depth: 200K-500K per chain D->Depth4 Mux1 Multiplex Level: Low (12-24 plex) Depth1->Mux1 End Finalized Experimental Design Mux1->End Mux2 Multiplex Level: Medium (48-96 plex) Depth2->Mux2 Mux2->End Mux3 Multiplex Level: High (96-192+ plex) Depth3->Mux3 Mux3->End Mux4 Multiplex Level: Chain-Specific (24-48 plex) Depth4->Mux4 Mux4->End

Diagram Title: Decision Logic for Multiplexing & Depth

G SamplePrep Sample RNA/DNA (96 Samples) Amp AmpliSeq PCR with Gene-Specific Primers and UMIs SamplePrep->Amp Lib Library Prep: Partial Adapter Addition, Index PCR (UDIs) Amp->Lib Pool Normalize & Pool Libraries Based on Multiplexing Design Lib->Pool Seq Sequencing (NovaSeq Flow Cell) Pool->Seq Data1 Demultiplex by Unique Dual Indexes Seq->Data1 Data2 UMI Clustering & Error Correction Data1->Data2 Data3 V(D)J Alignment & Clonotype Calling Data2->Data3 Output High-Resolution Repertoire Data Data3->Output DepthNode Key Optimization Step: Adjust Pooling Ratios for Target Depth DepthNode->Pool  Controls

Diagram Title: Optimized AmpliSeq Immune Repertoire Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Optimized Immune Repertoire Sequencing

Item Function & Role in Optimization
AmpliSeq for Illumina Immune Repertoire Panel (TCR Beta, IgH, etc.) Targeted primer panels for highly multiplexed PCR amplification of V(D)J regions from RNA/DNA, ensuring uniform coverage and minimizing bias.
AmpliSeq for Illumina PLUS Panel Extended panels for longer amplicons, enabling paired heavy/light chain (BCR) or alpha/beta chain (TCR) analysis from single cells or bulk RNA.
Unique Dual Indexes (UDI) Sets (e.g., IDT for Illumina) Allows high-level multiplexing (384+, 1536+) with minimal index hopping, critical for accurately assigning millions of reads to many samples.
Unique Molecular Identifiers (UMIs) Short random nucleotide tags incorporated during reverse transcription or early PCR cycles. Enables bioinformatic error correction and precise molecule counting, improving accuracy at lower sequencing depths.
High-Fidelity DNA Polymerase (e.g., Platinum SuperFi II) Crucial for accurate amplification of hyper-diverse repertoires with minimal PCR error, preserving sequence fidelity for clonal tracking.
Magnetic Bead-Based Cleanup Systems (e.g., AMPure XP) For consistent size selection and purification of amplicon libraries, removing primer dimers that consume sequencing depth.
Qubit Fluorometer & dsDNA HS Assay Kit Accurate quantification of library concentration for precise equimolar pooling, ensuring even depth across multiplexed samples.
Bioanalyzer/TapeStation (High Sensitivity DNA Kit) Quality control of final library fragment size distribution, confirming successful amplification of the target V(D)J regions.

Introduction Within the context of AmpliSeq for Illumina immune repertoire sequencing, raw sequencing data is inundated with technical noise. This document provides application notes and detailed protocols for a three-stage bioinformatic pipeline essential for deriving accurate, biologically relevant T-cell receptor (TCR) or B-cell receptor (BCR) clonality metrics. Effective filtering is a prerequisite for valid conclusions in vaccine, autoimmunity, and oncology drug development research.

Stage 1: Removing Artifacts and Contaminants Artifacts arise from sequencing errors, index hopping, and environmental contamination. The primary strategy involves filtering against control samples and quality thresholds.

Protocol 1.1: In-silico Contamination Filtering

  • Method: Align all reads to a composite reference (e.g., GRCh38 + phiX174). Use a k-mer-based classifier (Kraken2) against a standard microbial database.
  • Procedure:
    • Concatenate raw FASTQ files per sample.
    • Run Kraken2 with default parameters and the "Standard" database.
    • Parse the classification output. Discard reads classified under taxonomic IDs not belonging to the host (e.g., Homo sapiens) or expected vectors.
    • Retain only host-classified and unclassified reads for downstream analysis.
  • Key Metric: Typically, >95% of reads should be classified as host in a well-controlled experiment.

Protocol 1.2: Low-Quality and Chimeric Read Removal

  • Method: Use DADA2 or FASTP for quality control and chimera removal.
  • Procedure:
    • FASTP: Execute with parameters --detect_adapter_for_pe, --trim_poly_g, --length_required 50.
    • DADA2 (for single-end Amplicons): Filter with filterAndTrim(): maxN=0, maxEE=2.0, truncQ=2.
    • Chimera Removal: In DADA2, apply removeBimeraDenovo(method="consensus") on the amplicon sequence variant (ASV) table.

Data Summary Table 1: Artifact Removal Impact

Filtering Step Input Reads Output Reads % Retained Typical Reagent/Kit Source
Raw Demultiplexed Data 3,000,000 3,000,000 100% Illumina MiSeq Reagent Kit v3
Post Quality-Trim (FASTP) 3,000,000 2,820,000 94.0% N/A
Post Contamination Filter 2,820,000 2,774,000 98.4% N/A
Post Chimera Removal 2,774,000 2,220,000 80.0% N/A

Stage 2: Correcting PCR and Sequencing Errors PCR errors introduce artificial diversity. This stage collapses erroneous reads into true biological templates.

Protocol 2.1: Clustering-Based Error Correction

  • Method: Utilize a clustering tool like usearch or CD-HIT with a high-identity threshold.
  • Procedure:
    • Dereplicate quality-filtered reads.
    • Cluster sequences at 99% identity using the -cluster_fast command in usearch.
    • Designate the most abundant sequence in each cluster as the "true" template.
    • Sum the reads of all sequences within a cluster to the template count.

Protocol 2.2: Statistical Error Correction (MiXCR)

  • Method: Employ the MiXCR toolkit's built-in error-aware aligner.
  • Procedure:
    • Run the mixcr analyze amplicon command with the --illumina preset.
    • The algorithm aligns reads to V/D/J gene references, applying a quality-aware statistical model to distinguish true mutations from sequencing errors during the assembly of clonotypes.

Data Summary Table 2: Error Correction Efficacy

Method Pre-Correction Unique Sequences Post-Correction Unique Sequences Reduction Factor
Raw ASVs (DADA2) 85,000 N/A N/A
Clustering (CD-HIT 99%) 85,000 28,000 3.0x
Statistical (MiXCR) N/A (raw reads) 22,500 N/A

Stage 3: Normalizing for Clonality Metrics Post-filtering, read counts require normalization for meaningful inter-sample comparison of clonality.

Protocol 3.1: Down-Sampling to Equal Sequencing Depth

  • Method: Use rarefaction to a standardized number of reads per sample.
  • Procedure:
    • Calculate the minimum total productive reads across all samples in the cohort.
    • Using the R package vegan, execute the rrarefy() function on the clonotype count table, setting the sample size to the determined minimum.
    • Recalculate frequencies from the rarefied counts.

Protocol 3.2: Clonality Index Calculation

  • Method: Compute diversity indices on the normalized clonotype table.
  • Procedure:
    • Shannon Entropy (H): H = -sum(p_i * log(p_i)), where p_i is the frequency of clonotype i. Higher H indicates greater diversity.
    • Clonality (1 - Pielou's Evenness): Clonality = 1 - (H / log(S)), where S is the total number of unique clonotypes. Ranges from 0 (perfectly diverse) to 1 (monoclonal).
    • Gini-Simpson Index: 1 - sum(p_i^2). Probability two randomly selected reads are from different clonotypes.

Data Summary Table 3: Normalized Clonality Metrics

Sample Total Productive Reads Normalized Reads Unique Clonotypes (S) Shannon Entropy (H) Clonality (1-E)
Healthy 1 150,000 100,000 12,500 8.1 0.18
Healthy 2 180,000 100,000 11,800 7.9 0.20
Tumor 1 95,000 95,000 850 4.5 0.54
Tumor 2 220,000 100,000 1,200 5.1 0.49

The Scientist's Toolkit: Research Reagent & Software Solutions

Item Function
Illumina TCR/BCR Panel Target-specific primers for multiplex amplification of rearranged V(D)J regions.
MiXCR Software Integrated pipeline for alignment, error correction, and clonotype assembly.
DADA2 R Package Model-based inference of exact amplicon sequence variants (ASVs) from Illumina data.
FASTP Ultra-fast all-in-one FASTQ preprocessor for quality control and adapter trimming.
Kraken2 & Standard DB Rapid taxonomic classification of sequence reads to identify and remove contaminating sequences.
USearch/CD-HIT Algorithms for clustering and dereplicating nucleotide sequences to correct PCR errors.
vegan R Package Provides functions for ecological diversity analysis, including rarefaction and index calculation.

Diagram 1: Three-Stage Bioinformatic Workflow

G Raw Raw FASTQ Files S1 Stage 1: Artifact Removal Raw->S1 S2 Stage 2: Error Correction S1->S2 Sub1 Quality Trimming Contaminant Filter Chimera Removal S1->Sub1 S3 Stage 3: Normalization S2->S3 Sub2 Clustering or Statistical Correction S2->Sub2 Metrics Clonality Metrics S3->Metrics Sub3 Down-sampling Diversity Index Calculation S3->Sub3

Diagram 2: PCR Error Correction Logic

G PCR PCR Amplification (True Template + Errors) Seq Sequencing Read Pool PCR->Seq Cluster Cluster at 99% Identity Seq->Cluster Model Statistical Error Model Seq->Model Out1 Corrected Clonotype Table Cluster->Out1 Abundance-based consensus Out2 Corrected Clonotype Table Model->Out2 Likelihood-based assignment

Benchmarking Performance: Validation Metrics and How AmpliSeq Stacks Up to Other Methods

Within the broader thesis on AmpliSeq for Illumina immune repertoire (IR) panel research, establishing robust validation parameters is critical for generating reliable, publication-ready data. This application note details the experimental protocols and analytical frameworks required to validate IR sequencing assays, focusing on four pillars: Sensitivity, Reproducibility, Clonotype Detection Limit, and Quantitative Accuracy. These parameters ensure that observed clonal dynamics reflect true biology, enabling confident application in therapeutic antibody discovery, vaccine development, and biomarker identification.

Experimental Protocols for Key Validation Experiments

Protocol 2.1: Determining Sensitivity and Clonotype Detection Limit

Objective: To establish the minimum input copy number of a T-cell or B-cell receptor required for consistent detection and to define the limit of detection (LOD) for individual clonotypes. Materials: Synthetic TCR/BCR control templates (e.g., from Invitrogen Immune Sequencing Standards), mononuclear cells, AmpliSeq for Illumina Immune Repertoire Panel (e.g., TCR Beta Panel), Ion Chef System, Ion S5/Illumina MiSeq. Procedure:

  • Serial Dilution Series: Prepare a dilution series of synthetic immune receptor RNA or DNA standards with known clonotypes across a range from 10^6 to 10 copies per reaction.
  • Spike-in Experiment: Spike known, low-frequency clonotypes (e.g., 0.01%, 0.1%, 1%) into a background of polyclonal PBMC cDNA.
  • Library Preparation: Process all samples using the AmpliSeq Library Kit Plus and the targeted immune repertoire panel according to manufacturer's instructions.
  • Sequencing: Run on appropriate platform with sufficient depth (>5M reads per sample for high sensitivity).
  • Analysis: Use the ImmunoSEQ Analyzer or MiXCR software. The LOD is defined as the lowest input copy number at which a clonotype is detected with ≥95% probability. The Clonotype Detection Limit is reported as the smallest frequency (%) reliably quantifiable above background.

Protocol 2.2: Assessing Reproducibility

Objective: To evaluate technical precision across replicates, operators, and instrument runs. Procedure:

  • Replicate Design: Prepare ≥3 technical replicates from the same cDNA sample. Include inter-day and inter-operator replicates.
  • Full-Process Replication: Carry each replicate independently through the entire workflow: amplification, library preparation, templating, sequencing, and analysis.
  • Data Comparison: Calculate pairwise correlations (Pearson's r) of clonotype frequencies and the relative standard deviation (RSD%) for high-, medium-, and low-abundance clonotypes across replicates.

Protocol 2.3: Validating Quantitative Accuracy

Objective: To determine the linearity and accuracy of frequency measurements across the dynamic range. Procedure:

  • Artificial Repertoire Mixtures: Create benchmark samples by mixing cDNA from two or more distinct, well-characterized samples (e.g., a dominant clonotype cell line with polyclonal PBMCs) in defined proportions (e.g., 50%, 10%, 1%, 0.1%).
  • Sequencing & Analysis: Process mixtures and individual components. Compare the measured frequency of each "spiked" clonotype in the mixture to its expected frequency based on input proportions.
  • Linearity Assessment: Perform linear regression analysis (expected vs. observed frequency). Quantitative accuracy is reported as the slope (ideally 1.0) and R² value of the regression line.

Table 1: Typical Validation Performance for AmpliSeq-based Immune Repertoire Sequencing

Validation Parameter Target Performance Metric Typical Result (TCR Beta Panel) Key Influencing Factors
Sensitivity (Input) Minimum cell number for repertoire capture Reliable profiling from <10,000 cells (<100 ng RNA) RNA quality, PCR cycles, primer efficiency
Clonotype Detection Limit Lowest frequency reliably quantified 0.01% - 0.1% of total repertoire Sequencing depth, background noise, analysis pipeline
Quantitative Accuracy (Linearity) R² of expected vs. observed frequency R² ≥ 0.99 across 4 orders of magnitude Pipetting accuracy, primer bias, normalization
Reproducibility (Technical Replicates) Coefficient of Variation (CV) for high-abundance clonotypes CV < 5% for clonotypes >1% Library prep consistency, sequencing loading

Table 2: Comparison of Key Software Tools for Validation Analysis

Tool Primary Use in Validation Key Output Metric Link to Protocol
ImmunoSEQ Analyzer LOD calculation, reproducibility analysis Detection probability, frequency RSD 2.1, 2.2
MiXCR Clonotype tracking across replicates Clonotype overlap metrics (Jaccard index) 2.2
R/Bioconductor (e.g., vegan) Diversity index correlation analysis Spearman's rho for Shannon/Simpson indices 2.2, 2.3
Custom Python/R Scripts Linear regression for accuracy Slope, R², confidence intervals 2.3

Visualizations of Workflows and Relationships

validation_workflow start Sample Input (PBMCs/cDNA) dil Dilution/Spike-in (Protocol 2.1) start->dil prep AmpliSeq Library Preparation dil->prep seq NGS Sequencing (Illumina/Ion) prep->seq ana Bioinformatic Analysis seq->ana sens Sensitivity & LOD Report ana->sens Compare to input truth rep Reproducibility Metrics ana->rep Compare across replicates acc Quantitative Accuracy Report ana->acc Expected vs. Observed

Title: Immune Repertoire Validation Experimental Workflow

parameter_relationships panel AmpliSeq Panel Design sens Sensitivity panel->sens lod Clonotype Detection Limit panel->lod wet Wet-lab Protocol wet->sens rep Reproducibility wet->rep acc Quantitative Accuracy wet->acc seq Sequencing Depth & Quality seq->lod seq->rep seq->acc bio Bioinformatic Pipeline bio->lod bio->rep bio->acc lod->sens informs

Title: Key Parameters and Their Influencing Factors

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Immune Repertoire Validation Studies

Item Vendor (Example) Function in Validation
AmpliSeq for Illumina Immune Repertoire Panel (TCR Beta, IgH) Illumina Targeted multiplex PCR primer pools for specific receptor loci.
Ion AmpliSeq Library Kit 2.0 Thermo Fisher Core reagents for amplicon library construction and barcoding.
Synthetic Immune Receptor RNA/DNA Standards Invitrogen, IDT Defined clonotype mixtures for spike-in controls to establish LOD/accuracy.
Peripheral Blood Mononuclear Cells (PBMCs) Commercial Biobanks Provides polyclonal background for spike-in experiments.
High-Fidelity DNA Polymerase (e.g., Platinum SuperFi II) Thermo Fisher Reduces PCR error rates, critical for accurate clonotype calling.
Magnetic Bead Clean-up Kits (AMPure XP) Beckman Coulter For consistent size selection and purification post-amplification.
Qubit Fluorometer & dsDNA HS Assay Kit Thermo Fisher Accurate quantification of library yield pre-sequencing.
Bioanalyzer/TapeStation DNA Kits Agilent Assess library fragment size distribution and quality.
ImmunoSEQ Analyzer or MiXCR Software Adaptive Biotechnologies / Public Specialized bioinformatic pipelines for clonotype identification and quantification.

In the context of advancing research on AmpliSeq for Illumina immune repertoire panels, selecting the optimal methodology for immune receptor discovery is critical. This analysis compares the targeted AmpliSeq approach with broad, unbiased Whole Transcriptome RNA-Seq, detailing their applications in profiling T-cell receptors (TCR) and B-cell receptors (BCR).

Table 1: Core Technical and Performance Comparison

Feature AmpliSeq for Immune Repertoire (e.g., TCR/BCR Panels) Whole Transcriptome RNA-Seq
Primary Goal Targeted, high-depth sequencing of rearranged immune receptor loci (CDR3 regions). Unbiased profiling of the entire transcriptome, including immune transcripts.
Target Region Specific V(D)J gene segments. All polyadenylated RNA or total RNA.
Starting Input Low input compatible (10-100 ng total RNA or cDNA). Typically requires 100 ng - 1 µg total RNA for standard libraries.
Read Depth Required High depth per sample (5M+ reads) for rare clonotype detection. Lower depth per sample for expression (20-50M reads), but more for repertoire.
Focus on Isotypes Designed for specific isotype analysis (e.g., IgG, IgA). Can infer isotypes from constant region reads, but less direct.
Multiplexing Capacity High; designed for multiplexed PCR from many samples. Moderate; limited by barcoding during library prep.
Key Strength Superior sensitivity for rare clonotypes; quantitative for frequency. Discovery of novel V(D)J combinations; full transcriptome context.
Key Limitation Primer bias potential; limited to known, targeted regions. Inefficient sequencing depth on immune loci; higher cost for equivalent depth.
Optimal For Quantitative immune monitoring, minimal residual disease, vaccine response. Exploratory discovery, novel receptor identification, differential gene expression.

Table 2: Typical Output Metrics from a Representative Study

Metric AmpliSeq Immune Panel Whole Transcriptome RNA-Seq (RepSeq Analysis)
% Reads On-Target >80% <5% (on immune receptor loci)
Clonotypes Detected (per sample) 50,000 - 200,000+ 1,000 - 10,000 (without enrichment)
Effective Depth on TCR/BCR ~4-5 million reads ~50,000-500,000 reads (from 50M WTS reads)
Sample-to-Result Time (Hands-on) ~1.5 days ~3-4 days
Cost per Sample (Seq. Only) $$ $$$$ (to achieve comparable immune locus depth)

Experimental Protocols

Protocol 1: Immune Repertoire Profiling Using AmpliSeq for Illumina

This protocol details library preparation for targeted immune receptor sequencing.

Materials: See "Research Reagent Solutions" table. Procedure:

  • RNA Isolation & QC: Extract total RNA from PBMCs or tissue (e.g., using TRIzol). Assess integrity (RIN > 7) and quantify.
  • cDNA Synthesis: Perform reverse transcription using a high-fidelity reverse transcriptase and gene-specific primers targeting constant regions of TCR/BCR transcripts.
  • Targeted PCR Amplification:
    • Utilize the commercial AmpliSeq immune panel kit (e.g., AmpliSeq for Illumina TCR Beta-SR Assay).
    • In a multiplex PCR, amplify rearranged V(D)J regions using a pool of primers covering all known V and J gene segments.
    • Cycling conditions: Initial denaturation at 95°C for 2 min; 20-25 cycles of (95°C for 15s, 60°C for 4 min); final extension at 60°C for 1 min.
  • Partial Digestion of Primer Sequences: Add a digestion mix to partially remove the introduced primer sequences. Incubate at 37°C for 10 min.
  • Adapter Ligation: Ligate Illumina P5 and P7-compatible adapters, containing sample-specific barcodes (indexes), to the amplicons.
  • Library Purification & QC: Purify libraries using magnetic beads. Assess library size (~300-350 bp) and concentration via capillary electrophoresis.
  • Normalization, Pooling, and Sequencing: Normalize libraries, pool equimolarly, and sequence on an Illumina platform (e.g., MiSeq, NextSeq) with a 2x150 bp or 2x300 bp run to achieve >5 million paired-end reads per sample.
  • Data Analysis: Use dedicated software (e.g., MiXCR, ImmunoSEQ Analyzer) for alignment to V(D)J reference, clonotype assembly, and diversity analysis.

Protocol 2: Immune Receptor Discovery from Whole Transcriptome RNA-Seq Data

This protocol describes how to extract and analyze immune repertoire information from standard RNA-seq data.

Materials: See "Research Reagent Solutions" table. Procedure:

  • Library Preparation: Prepare standard Illumina RNA-seq libraries using a poly-A selection or ribosomal RNA depletion kit, following the manufacturer's protocol. Include unique dual indexes (UDIs) for sample multiplexing.
  • Sequencing: Sequence the library pool on an Illumina NovaSeq or HiSeq platform to a depth of at least 50 million paired-end reads per sample (100-150 bp read length recommended).
  • Primary Transcriptomic Analysis: Align reads to the human reference genome (e.g., GRCh38) using a splice-aware aligner (e.g., STAR). Perform gene-level quantification.
  • Immune Receptor Read Extraction:
    • Extract reads that did not align, or aligned poorly, to the standard reference genome.
    • Alternatively, align all reads directly to a curated set of V, D, J, and C gene reference sequences from the IMGT database using a specialized tool.
  • Clonotype Assembly & Analysis:
    • Process extracted reads using immune repertoire-specific tools (e.g., TRUST4, BRACER, VDJPuzzle).
    • Tools will identify CDR3 sequences, assign V(D)J genes, and output clonotype frequency tables.
  • Integrative Analysis: Correlate clonotype abundance and diversity metrics with host gene expression data from Step 3 to link immune receptor dynamics with transcriptional states.

Visualizations

workflow_choice Start Research Question: Immune Receptor Analysis Q1 Primary Goal: Quantification or Discovery? Start->Q1 Q2 Sample Input Limited or Abundant? Q1->Q2 Quantify known clonotypes WTS Choose Whole Transcriptome Q1->WTS Discover novel variants/genes Q3 Budget for Sequencing Depth? Q2->Q3 Abundant Amp Choose AmpliSeq Q2->Amp Limited (e.g., FFPE, rare cells) Q3->Amp Focus budget on immune loci depth Q3->WTS Budget for broad coverage & context

Title: Decision Workflow for Immune Receptor Profiling Method Selection

protocol_flow cluster_ampliseq AmpliSeq Protocol cluster_rnaseq Whole Transcriptome Protocol A1 Total RNA (10-100 ng) A2 cDNA Synthesis (Constant Region Primers) A1->A2 A3 Multiplex PCR (V/J Primer Panels) A2->A3 A4 Partial Digest & Adapter Ligation A3->A4 A5 Sequence (2x150 bp, 5M+ reads) A4->A5 A6 Targeted Analysis: Clonotype Frequency A5->A6 R1 Total RNA (100 ng - 1 µg) R2 Poly-A Selection or rRNA Depletion R1->R2 R3 Library Prep & Adapter Ligation R2->R3 R4 Sequence (2x100 bp, 50M+ reads) R3->R4 R5 Genome Alignment & Expression Quant R4->R5 R6 Specialized Repertoire Extraction R4->R6

Title: AmpliSeq vs Whole Transcriptome Experimental Workflows

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Immune Repertoire Studies

Item Function Example Product/Category
Total RNA Isolation Kit High-yield, high-integrity RNA extraction from cells/tissue. TRIzol/column-based kits (Qiagen, Thermo Fisher).
Targeted Immune Panel Kit All-in-one solution for AmpliSeq library prep from cDNA. AmpliSeq for Illumina Immune Repertoire Plus (TCR/BCR).
Whole Transcriptome Kit Library prep for total or mRNA-seq. TruSeq Stranded mRNA, Illumina RNA Prep with Enrichment.
High-Fidelity RT Enzyme Accurate cDNA synthesis from RNA template. SuperScript IV, Maxima H Minus.
Universal PCR Master Mix Robust, high-fidelity multiplex PCR amplification. Platinum SuperFi II, AmpliSeq Mix.
SPR/Bead Purification Beads Size selection and cleanup of DNA libraries. AMPure XP Beads.
Library QC System Accurate sizing and quantification of final libraries. Agilent Bioanalyzer/TapeStation, Qubit Fluorometer.
Dedicated Analysis Software Align sequences, call clonotypes, and calculate diversity. MiXCR, ImmunoSEQ Analyzer, TRUST4, VDJPuzzle.
IMGT Database The international reference for immunoglobulin and TCR genetics. Critical resource for V(D)J gene annotation.

This Application Note, framed within a broader thesis on AmpliSeq for Illumina immune repertoire research, provides a comparative analysis of targeted sequencing approaches for profiling adaptive immune receptor repertoires. The analysis focuses on AmpliSeq for Illumina, multiplex PCR-based methods, and RACE-based techniques, with specific consideration of molecular barcoding (Unique Molecular Identifiers, UMIs) integration. Accurate profiling of B-cell and T-cell receptor diversity is critical for vaccine development, oncology biomarker discovery, and autoimmune disease research.

Table 1: Core Technology Comparison

Feature AmpliSeq for Illumina Multiplex PCR (Conventional) RACE-based (e.g., SMARTer)
Principle Highly multiplexed, primer pool-based PCR (amplicon) Multiple primer pairs in single tube cDNA end extension with common primer sequence
Starting Material RNA (≥20 ng) or DNA RNA (often requires >100 ng) RNA (can work with low input, ~1 ng)
Primer Design Large, optimized pool of target-specific primers (hundreds to thousands) Limited set of gene-specific primers Universal primer on poly-A tail; gene-specific primer on constant region
Target Region Predetermined V(D)J segments Predetermined V(D)J segments Full-length V(D)J transcript
Bias Risk Medium (optimized pools reduce, but do not eliminate, bias) High (primer competition & annealing efficiency variance) Low (avoids V-gene primer bias)
Throughput High (highly multiplexed, automated) Medium Low to Medium
Molecular Barcoding (UMI) Integration Compatible (with specific UMI adapters, e.g., from iSeq, NovaSeq) Can be added but requires custom design Commonly integrated (e.g., in 5' RACE oligo)
Primary Application High-throughput, standardized immune profiling Targeted studies of known V genes Discovery-focused, full-length repertoire analysis
Approximate Cost per Sample (Reagents) $80 - $150 $30 - $80 $100 - $200

Table 2: Performance Metrics from Recent Studies (2023-2024)

Metric AmpliSeq Immune Repertoire Panel Multiplex PCR (5' RACE with UMIs) Hybrid Capture-Based
Clonotype Detection Sensitivity >99% for covered targets ~95% (limited by 5' primer efficiency) >99.5% (with baits covering all loci)
Reproducibility (Pearson R²) 0.98 - 0.99 0.90 - 0.95 0.97 - 0.99
PCR Duplication Rate (without UMIs) 15-25% 20-40% 5-15% (library prep dependent)
PCR Duplication Rate (with UMIs) <2% <5% <1%
Input RNA Recommendation 10-100 ng 50-1000 ng 10-1000 ng
Typical Time from Sample to Data 1.5 - 2.5 days 2 - 3 days 3 - 5 days

Detailed Protocols

Protocol 3.1: AmpliSeq for Illumina Immune Repertoire Workflow with UMIs

Objective: Generate immune repertoire sequencing libraries from human RNA using the AmpliSeq Immune Repertoire Panel with integrated Unique Molecular Identifiers (UMIs) for error correction and accurate clonotype quantification. Materials: AmpliSeq Immune Repertoire Panel (Human BCR or TCR), Ion Total RNA-Seq Kit v2, Ion Xpress Barcode Adapters (with UMIs), Agencourt AMPure XP Beads, High-Sensitivity DNA Kit (Bioanalyzer). Procedure:

  • RNA QC: Assess RNA integrity (RIN > 7.0) using a Bioanalyzer.
  • Reverse Transcription: Convert 10-100 ng total RNA to cDNA using the included kit with gene-specific primers from the panel.
  • Target Amplification: Perform multiplex PCR using the large, pre-designed primer pool (AmpliSeq panel). Cycle conditions: 99°C for 2 min; [99°C for 15 sec, 60°C for 4 min] x 25 cycles; 10°C hold.
  • Partial Digestion: Treat amplicons with FuPa reagent to partially digest primer sequences and phosphorylate ends.
  • Adapter Ligation: Ligate Ion Xpress Barcode Adapters (containing sample barcodes and UMIs) to digested amplicons. Clean up with AMPure XP beads.
  • Library Amplification: Amplify the ligated library for 5-7 cycles. Perform final bead clean-up.
  • Library QC: Quantify using Qubit and assess size distribution (~300 bp) via Bioanalyzer High-Sensitivity DNA chip.
  • Template Preparation & Sequencing: Proceed to Ion Chef / Ion GeneStudio S5 or Illumina platform-specific preparation (following manufacturer's cross-compatibility guidelines).

Protocol 3.2: Multiplex PCR with Molecular Barcoding (Two-Step)

Objective: Generate immune repertoire libraries using a custom multiplex PCR approach incorporating UMIs at the RT step. Materials: SuperScript IV Reverse Transcriptase, Template Switching Oligo (TSO) with UMI, Isothermal Amplification Buffer (e.g., from SMARTer), Multiplex V-gene primers, High-Fidelity DNA Polymerase, AMPure XP Beads. Procedure:

  • Primer Design: Design multiplex primer sets for V gene families. Include a common linker sequence on the 5' end of each primer.
  • Reverse Transcription with UMI/TSO: To RNA, add V-gene multiplex primers and TSO containing a UMI. Perform RT with template switching capability.
  • cDNA Amplification: Perform limited-cycle (10-12 cycles) PCR using a primer complementary to the TSO sequence and a primer for the common linker on the V-gene primers.
  • Nested PCR for Sequencing Adapters: Use a second, nested PCR (15 cycles) with primers containing full Illumina adapter sequences (P5/P7) and sample indices. Clean up with AMPure XP beads.
  • QC & Sequencing: Quantify and size-select libraries (e.g., 2% agarose gel or bead-based size selection). Sequence on Illumina MiSeq or HiSeq.

Protocol 3.3: 5' RACE-based Full-Length Repertoire Profiling

Objective: Capture complete, unbiased V(D)J transcripts from low-input RNA samples. Materials: SMARTer Human BCR/TCR Profiling Kit (Takara Bio), Advantage 2 PCR Kit, AMPure XP Beads. Procedure:

  • 5' RACE cDNA Synthesis: Use a primer annealing to the constant region (C-region) of the target receptor mRNA. The reverse transcriptase adds nontemplated nucleotides upon reaching the 5' end, to which a "template-switch" oligo (TSO) anneals, creating a common 5' sequence for all transcripts.
  • cDNA Amplification: Perform LD-PCR using a primer targeting the TSO sequence and a primer targeting the C-region.
  • Index PCR for Illumina: Use the amplified cDNA as template in a second PCR with indexed primers that add full sequencing adapters.
  • Purification & Size Selection: Perform double-sided bead-based size selection to remove primer dimers and large contaminants.
  • Sequencing: Pool libraries and sequence on an Illumina platform (minimum 2x300 bp for full-length coverage).

Visualizations

workflow_ampliseq RNA Total RNA (RIN > 7) RT Reverse Transcription with Gene-Specific Primers RNA->RT Amp Multiplex Target Amplification (AmpliSeq) RT->Amp Dig Partial Digestion & Phosphorylation (FuPa) Amp->Dig Lig Adapter Ligation (Ion Xpress Barcode + UMI) Dig->Lig LibAmp Library Amplification (5-7 cycles) Lig->LibAmp QC Library QC (Qubit, Bioanalyzer) LibAmp->QC Seq Sequencing (Illumina/Ion) QC->Seq

Diagram Title: AmpliSeq Immune Repertoire Workflow with UMI Integration

tech_comparison cluster_0 Bias Risk cluster_1 Throughput cluster_2 Method High High Med Medium Low Low H High M Medium L Low/Medium MxPCR Multiplex PCR MxPCR->High MxPCR->M AmpSeq AmpliSeq AmpSeq->Med AmpSeq->H RACE RACE-based RACE->Low RACE->L

Diagram Title: Technology Comparison: Bias vs. Throughput

umi_correction Start Raw Sequencing Reads with UMIs Step1 1. Cluster Reads by Source UMI Sequence Start->Step1 Step2 2. Perform Multiple Sequence Alignment (MSA) per UMI Group Step1->Step2 Step3 3. Generate Consensus Sequence (Removes PCR & Sequencing Errors) Step2->Step3 Step4 4. Annotate Consensus Sequences (V(D)J Alignment, Clonotype Calling) Step3->Step4 Result Error-Corrected Clonotype Table Step4->Result

Diagram Title: UMI-Based Error Correction Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Immune Repertoire Sequencing

Item Vendor Example(s) Primary Function in Protocol
AmpliSeq Immune Repertoire Panel Thermo Fisher Scientific Provides the comprehensive, optimized primer pool for targeted amplification of human TCR/BCR loci.
Ion Xpress Barcode Adapters (with UMIs) Thermo Fisher Scientific Contains sample-specific barcodes and Unique Molecular Identifiers (UMIs) for multiplexing and error correction.
SMARTer Human TCR/BCR Profiling Kit Takara Bio Provides all reagents for 5' RACE-based, full-length repertoire construction from low-input RNA.
SuperScript IV Reverse Transcriptase Thermo Fisher Scientific High-temperature, high-fidelity RT enzyme for robust first-strand cDNA synthesis, especially for complex RNA.
KAPA HiFi HotStart ReadyMix Roche High-fidelity DNA polymerase for amplification steps where minimizing PCR errors is critical (e.g., UMI consensus building).
Agencourt AMPure XP Beads Beckman Coulter Solid-phase reversible immobilization (SPRI) beads for predictable and efficient DNA clean-up and size selection.
High Sensitivity DNA Kit Agilent Technologies Used with the Bioanalyzer instrument to accurately quantify and assess size distribution of final sequencing libraries.
Template Switching Oligo (TSO) Integrated DNA Technologies (IDT) Essential for 5' RACE methods; enables the addition of a universal sequence to the 5' end of cDNA during RT.
Multiplex PCR Primer Sets (Custom) IDT, Thermo Fisher User-defined primer mixes for specific V-gene targets; requires careful optimization to minimize amplification bias.

Within the broader thesis investigating AmpliSeq for Illumina immune repertoire analysis, selecting the appropriate panel and methodology requires a systematic evaluation of key performance parameters. This application note provides a detailed comparison of available AmpliSeq Immune Repertoire Panels, supported by experimental protocols and analytical workflows, to guide researchers, scientists, and drug development professionals in aligning platform capabilities with specific research objectives.

Comparative Performance Metrics

The choice of panel impacts core experimental outcomes. Quantitative data for major panels are summarized below.

Table 1: Comparison of AmpliSeq for Illumina Immune Repertoire Panels

Panel Name Target Loci Throughput (Samples per Run) Sensitivity (Input RNA) Approximate Cost per Sample (Reagents Only) Primary Analysis Software
AmpliSeq for Illumina Immune Repertoire - TCR Beta Human TRB Up to 384 (NovaSeq) 1-10 ng total RNA $$ Immune Repertoire (IR) Analyzer, MiXCR
AmpliSeq for Illumina B-Cell Receptor Human IGH, IGK, IGL Up to 96 (MiSeq) 10-100 ng total RNA $$$ IR Analyzer, Partis
AmpliSeq for Illumina Mouse B-Cell Receptor Mouse IGH, IGK, IGL Up to 96 (MiSeq) 10-100 ng total RNA $$$ IR Analyzer
AmpliSeq for Illumina Pan-Cancer 1385 genes + TRB, IGH 8-16 (iSeq) 10 ng total RNA $$$$ Local IR App (DRAGEN), Basespace

Note: Throughput is system-dependent. Cost is indicative and includes library prep & sequencing; $$ = $50-$100, $$$ = $100-$200, $$$$ = >$200.

Experimental Protocols

Protocol 1: Library Preparation using AmpliSeq Immune Repertoire Panels

Objective: Generate sequencing-ready libraries from RNA/cDNA samples. Materials: AmpliSeq Immune Repertoire Panel (specific to species/locus), AmpliSeq Library PLUS for Illumina, SuperScript IV Reverse Transcriptase, Qubit dsDNA HS Assay Kit, SPRISelect beads.

  • Reverse Transcription: Convert 1-100 ng total RNA (see Table 1) to cDNA using SuperScript IV with random hexamers.
  • Target Amplification: Perform multiplex PCR using the specific AmpliSeq primer pool. Cycle conditions: 99°C for 2 min; [98°C for 15 sec, 60°C for 4 min] x 25 cycles; 10°C hold.
  • Partial Digestion: Treat PCR products with FuPa reagent to partially digest primers and phosphorylate amplicons (30°C for 10 min, 50°C for 10 min, 55°C for 10 min, then hold at 10°C).
  • Adapter Ligation: Ligate Illumina-compatible barcoded adapters (from Library PLUS kit) to digested amplicons (22°C for 30 min, 68°C for 5 min, then hold at 10°C).
  • Post-Ligation Cleanup: Purify ligated products using SPRISelect beads (0.8x ratio).
  • Library Amplification: Enrich adapter-ligated DNA with limited-cycle PCR (72°C for 3 min; [98°C for 15 sec, 64°C for 1 min] x 5-7 cycles; 72°C for 1 min).
  • Final Purification & QC: Clean final library with SPRISelect beads (0.8x ratio). Quantify using Qubit dsDNA HS Assay and assess fragment size (~320-350bp) via bioanalyzer.

Protocol 2: Immune Repertoire Data Processing with the IR Analyzer

Objective: Process raw FASTQ files to clonotype tables.

  • Upload: Upload demultiplexed FASTQ files to the Illumina BaseSpace Sequence Hub.
  • Application Selection: Launch the "Immune Repertoire (IR) Analyzer" app.
  • Parameter Configuration:
    • Select correct Panel (e.g., TCR Beta).
    • Set Species (Human/Mouse).
    • Specify Analysis Type (e.g., Clonotype).
    • Enable Advanced collapsing for PCR/sequencing error correction.
  • Run Initiation: Start the analysis. The app executes read alignment (to IMGT references), CDR3 extraction, and clonotype collapsing.
  • Output Retrieval: Download results, including the primary clonotypes.csv file containing frequencies, CDR3 nucleotide/amino acid sequences, V/D/J gene assignments, and read counts.

Visualizations

workflow Start Total RNA (1-100 ng) RT Reverse Transcription & cDNA Synthesis Start->RT Amp Multiplex PCR with Panel-Specific Primers RT->Amp Digest FuPa Treatment (Partial Digest & Phosphorylation) Amp->Digest Ligate Adapter Ligation (Add Barcodes & Indices) Digest->Ligate Clean1 SPRI Bead Cleanup Ligate->Clean1 Enrich Library Amplification PCR Clean1->Enrich Clean2 SPRI Bead Cleanup Enrich->Clean2 QC Library QC (Qubit, Bioanalyzer) Clean2->QC Seq Illumina Sequencing QC->Seq Analyze IR Analyzer Processing Seq->Analyze

Title: AmpliSeq Immune Repertoire Library Prep and Analysis Workflow

logic Goal Primary Research Goal T High Throughput (e.g., Population Studies) Goal->T Prioritize S High Sensitivity (e.g., Low-Abundance Clones) Goal->S Prioritize C Low Cost per Sample Goal->C Prioritize E Streamlined, Integrated Analysis Goal->E Prioritize PanelT TCR Beta Panel T->PanelT PanelBCR BCR Panels S->PanelBCR C->PanelT PanelPan Pan-Cancer Panel E->PanelPan

Title: Panel Selection Logic Based on Prioritized Research Goal

The Scientist's Toolkit

Table 2: Essential Research Reagent Solutions for AmpliSeq Immune Repertoire Research

Item Function in Workflow
AmpliSeq Immune Repertoire Primer Panel Target-specific primer pools for multiplex amplification of TCR or BCR loci.
AmpliSeq Library PLUS for Illumina Provides enzymes, buffers, and indexed adapters for library construction.
SuperScript IV Reverse Transcriptase High-efficiency, thermostable reverse transcriptase for robust first-strand cDNA synthesis from RNA.
RNase Inhibitor Protects RNA templates from degradation during reverse transcription.
SPRISelect / AMPure XP Beads Solid-phase reversible immobilization beads for size selection and purification of DNA fragments.
Qubit dsDNA High Sensitivity (HS) Assay Fluorometric quantification of low-concentration DNA libraries, critical for accurate pooling.
Agilent High Sensitivity DNA Kit (Bioanalyzer) Electrophoretic analysis for assessing library fragment size distribution and quality.
Illumina Sequencing Kits (MiSeq, NextSeq, NovaSeq) System-specific reagent cartridges for cluster generation and sequencing-by-synthesis.

Conclusion

AmpliSeq for Illumina immune repertoire panels provides a robust, targeted, and highly sensitive solution for dissecting adaptive immune responses. By understanding its foundational principles, researchers can effectively apply the methodology to diverse fields from immuno-oncology to infectious disease. Adherence to optimized protocols and troubleshooting guidelines ensures generation of high-quality, reproducible data critical for meaningful biological interpretation. When validated and contextualized against alternative methods, AmpliSeq emerges as a powerful tool for focused, high-throughput immune receptor profiling. The future of this technology lies in its integration with single-cell analysis and spatial transcriptomics, promising even deeper insights into the cellular context of immune clonotypes. This will accelerate the discovery of predictive biomarkers, therapeutic targets, and personalized immunodiagnostics, fundamentally advancing translational research and clinical development.